Oligonucleotide compositions and methods for the modulation of the expression of B7 protein

ABSTRACT

Compositions and methods for the treatment of asthma with oligonucleotides which specifically hybridize with nucleic acids encoding B7 proteins.

INTRODUCTION

[0001] This is a continuation-in-part of U.S. application Ser. No.09/851,871, filed May 9, 2001, which is a continuation-in-part ofInternational Patent Application No. PCT/US00/14471, which is acontinuation-in-part of U.S. application Ser. No. 09/326,186, filed Jun.4, 1999, which is a continuation-in-part of U.S. application Ser. No.08/777,266, filed Dec. 31, 1996, the entire disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to diagnostics, research reagents andtherapeutics for disease states which respond to modulation of T cellactivation. In particular, this invention relates to antisenseoligonucleotide interactions with certain messenger ribonucleic acids(mRNAs) or DNAs involved in the synthesis of proteins that modulate Tcell activation. Antisense oligonucleotides designed to hybridize tonucleic acids encoding B7 proteins are provided. These oligonucleotideshave been found to lead to the modulation of the activity of the RNA orDNA, and thus to the modulation of T cell activation. Palliative,therapeutic and prophylactic effects result.

BACKGROUND OF THE INVENTION

[0003] Inflammation is a localized protective response mounted bytissues in response to injury, infection, or tissue destructionresulting in the destruction of the infectious or injurious agent andisolation of the injured tissue. A typical inflammatory responseproceeds as follows: recognition of an antigen as foreign or recognitionof tissue damage, synthesis and release of soluble inflammatorymediators, recruitment of inflammatory cells to the site of infection ortissue damage, destruction and removal of the invading organism ordamaged tissue, and deactivation of the system once the invadingorganism or damage has been resolved. In many human diseases with aninflammatory component, the normal, homeostatic mechanisms whichattenuate the inflammatory responses are defective, resulting in damageand destruction of normal tissue.

[0004] Cell-cell interactions are involved in the activation of theimmune response at each of the stages described above. One of theearliest detectable events in a normal inflammatory response is adhesionof leukocytes to the vascular endothelium, followed by migration ofleukocytes out of the vasculature to the site of infection or injury. Ingeneral, the first inflammatory cells to appear at the site ofinflammation are neutrophils, followed by monocytes and lymphocytes.Cell-cell interactions are also critical for activation of bothB-lymphocytes (B cells) and T-lymphocytes (T cells) with resultingenhanced humoral and cellular immune responses, respectively.

[0005] The hallmark of the immune system is its ability to distinguishbetween self (host) and nonself (foreign invaders). This remarkablespecificity exhibited by the immune system is mediated primarily by Tcells. T cells participate in the host's defense against infection butalso mediate organ damage of transplanted tissues and contribute to cellattack in graft-versus-host disease (GVHD) and some autoimmune diseases.In order to induce an antigen-specific immune response, a T cell mustreceive signals delivered by an antigen-presenting cell (APC). Tcell-APC interactions can be divided into three stages: cellularadhesion, T cell receptor (TCR) recognition, and costimulation. At leasttwo discrete signals are required from an APC for induction of T cellactivation. The first signal is antigen-specific and is provided whenthe TCR interacts with an antigen in the context of a majorhistocompatibility complex (MHC) protein, or an MHC-related CD1 protein,expressed on the surface of an APC (“CD,” standing for “cluster ofdifferentiation,” is a term used to denote different T cell surfacemolecules). The second (costimulatory) signal involves the interactionof the T cell surface antigen, CD28, with its ligand on the APC, whichis a member of the B7 family of proteins.

[0006] CD28, a disulfide-linked homodimer of a 44 kilodalton polypeptideand a member of the immunoglobulin superfamily, is one of the majorcostimulatory signal receptors on the surface of a resting T cell for Tcell activation and cytokine production (Allison, Curr. Opin. Immunol.,1994, 6, 414; Linsley and Ledbetter, Annu. Rev. Immunol., 1993, 11, 191;June et al., Immunol. Today, 1994, 15, 321). Signal transduction throughCD28 acts synergistically with TCR signal transduction to augment bothinterleukin-2 (IL-2) production and proliferation of naive T cells. B7-1(also known as CD80) was the first ligand identified for CD28 (Liu andLinsley, Curr. Opin. Immunol., 1992, 4, 265). B7-1 is normally expressedat low levels on APCs, however, it is upregulated following activationby cytokines or ligation of cell surface molecules such as CD40(Lenschow et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11054; Nabaviet al., Nature, 1992, 360, 266). Initial studies suggested that B7-1 wasthe CD28 ligand that mediated costimulation (Reiser et al., Proc. Natl.Acad. Sci. U.S.A., 1992, 89, 271; Wu et al., J. Exp. Med., 1993, 178,1789; Harlan et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 3137).However, the subsequent demonstration that anti-B7-1 monoclonalantibodies (mabs) had minimal effects on primary mixed lymphocytereactions and that B7-1-deficient mice responded normally to antigens(Lenschow et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11054;Freeman et al., Science, 1993, 262, 909) resulted in the discovery of asecond ligand for the CD28 receptor, B7-2 (also known as CD86). Incontrast with anti-B7-1 mAbs, anti-B7-2 mabs are potent inhibitors of Tcell proliferation and cytokine production (Wu et al., J. Exp. Med.,1993, 178, 1789; Chen et al., J. Immunol., 1994, 152, 2105; Lenschow etal., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11054). B7:CD28 signalingmay be a necessary component of other T cell costimulatory pathways,such as CD40:CD40L (CD40 ligand) signaling (Yang et al., Science, 1996,273, 1862).

[0007] In addition to binding CD28, B7-1 and B7-2 bind the cytolyticT-lymphocyte associated protein CTLA4. CTLA4 is a protein that isstructurally related to CD28 but is expressed on T cells only afteractivation (Linsley et al., J. Exp. Med., 1991, 174, 561). A solublerecombinant form of CTLA4, CTLA4-Ig, has been determined to be a moreefficient inhibitor of the B7:CD28 interaction than monoclonalantibodies directed against CD28 or a B7 protein. In vivo treatment withCTLA4-Ig results in the inhibition of antibody formation to sheep redblood cells or soluble antigen (Linsley et al., Science, 1992, 257,792), prolongation of cardiac allograft and pancreatic islet xenograftsurvival (Lin et al., J. Exp. Med., 1993, 178, 1801; Lenschow et al.,1992, Science, 257, 789; Lenschow et al., Curr. Opin. Immunol., 1991, 9,243), and significant suppression of immune responses in GVHD (Hakim etal., J. Immun., 1995, 155, 1760). It has been proposed that CD28 andCTLA4, although both acting through common B7 receptors, serve opposingcostimulatory and inhibitory functions, respectively (Allison et al.,Science, 1995, 270, 932). CTLA41 g, which binds both B7-1 and B7-2molecules on antigen-presenting cells, has been shown to block T-cellcostimulation in patients with stable psoriasis vulgaris, and to cause a50% or greater sustained improvement in clinical disease activity in 46%of the patients to which it was administered. This result wasdose-dependent. Abrams et al., J. Clin. Invest., 1999, 9, 1243-1225.

[0008] European Patent Application No. EP 0 600 591 discloses a methodof inhibiting tumor cell growth in which tumor cells from a patient arerecombinantly engineered ex vivo to express a B7-1 protein and thenreintroduced into a patient. As a result, an immunologic response isstimulated against both B7-transfected and nontransfected tumor cells.

[0009] International Publication No. WO95/03408 discloses nucleic acidsencoding novel CTLA4/CD28 ligands which costimulate T cell activation,including B7-2 proteins. Also disclosed are antibodies to B7-2 proteinsand methods of producing B7-2 proteins.

[0010] International Publication No. WO95/05464 discloses a polypeptide,other than B7-1, that binds to CTLA4, CD28 or CTLA4-Ig. Also disclosedare methods for obtaining a nucleic acid encoding such a polypeptide.

[0011] International Publication No. WO 95/06738 discloses nucleic acidsencoding B7-2 (also known as B70) proteins. Also disclosed areantibodies to B7-2 proteins and methods of producing B7-2 proteins.

[0012] European Patent Application No. EP 0 643 077 discloses amonoclonal antibody which specifically binds a B7-2 (also known as B70)protein. Also disclosed are methods of producing monoclonal antibodieswhich specifically bind a B7-2 protein.

[0013] U.S. Pat. No. 5,434,131 discloses the CTLA4 protein as a ligandfor B7 proteins. Also disclosed are methods of producing CTLA4 fusionproteins (e.g., CTLA4-Ig) and methods of regulating immune responsesusing antibodies to B7 proteins or CTLA4 proteins.

[0014] International Publication No. WO95/22619 discloses antibodiesspecific to B7-1 proteins which do not bind to B7-2 proteins. Alsodisclosed are methods of regulating immune responses using antibodies toB7-1 proteins.

[0015] International Publication No. WO95/34320 discloses methods forinhibiting T cell responses using a first agent which inhibits acostimulatory agent, such as an CTLA4-Ig fusion protein, and a secondagent which inhibits cellular adhesion, such as an anti-LFA-1 antibody.Such methods are indicated to be particularly useful for inhibiting therejection of transplanted tissues or organs.

[0016] International Publication No. WO95/32734 discloses Fc RIIbridging agents which either prevent the upregulation of B7 molecules orimpair the expression of ICAM-3 on antigen presenting cells. Such FcRIIbridging agents include proteins such as aggregated human IgG moleculesor aggregated Fc fragments of human IgG molecules.

[0017] International Publication No. WO96/11279 discloses recombinantviruses comprising genetic sequences encoding (1) one or moreimmunostimulatory agents, including B7-1 and B7-2, and (2) and antigensfrom a disease causing agent. Also disclosed are methods of treatingdiseases using such recombinant viruses.

[0018] To date, there are no known therapeutic agents which effectivelyregulate and prevent the expression of B7 proteins such as B7-1 andB7-2. Thus, there is a long-felt need for compounds and methods whicheffectively modulate critical costimulatory molecules such as the B7proteins.

SUMMARY OF THE INVENTION

[0019] In accordance with the present invention, oligonucleotides areprovided which specifically hybridize with nucleic acids encoding B7-1or B7-2. Certain oligonucleotides of the invention are designed to bindeither directly to mRNA transcribed from, or to a selected DNA portionof, the B7-1 or B7-2 gene, thereby modulating the amount of proteintranslated from a B7-1 or B7-2 mRNA or the amount of mRNA transcribedfrom a B7-1 or B7-2 gene, respectively.

[0020] Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides are commonly described as“antisense.” Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents.

[0021] It has been discovered that the B7-1 and B7-2 genes, encodingB7-1 and B7-2 proteins, respectively, are particularly amenable to thisapproach. As a consequence of the association between B7 expression andT cell activation and proliferation, inhibition of the expression ofB7-1 or B7-2 leads to inhibition of the synthesis of B7-1 or B7-2,respectively, and thereby inhibition of T cell activation andproliferation. Additionally, the oligonucleotides of the invention maybe used to inhibit the expression of one of several alternativelyspliced mRNAs of a B7 transcript, resulting in the enhanced expressionof other alternatively spliced B7 mRNAs. Such modulation is desirablefor treating various inflammatory or autoimmune disorders or diseases,or disorders or diseases with an inflammatory component such as asthma,juvenile diabetes mellitus, myasthenia gravis, Graves' disease,rheumatoid arthritis, allograft rejection, inflammatory bowel disease,multiple sclerosis, psoriasis, lupus erythematosus, systemic lupuserythematosus, diabetes, multiple sclerosis, contact dermatitis,rhinitis, various allergies, and cancers and their metastases. Suchmodulation is further desirable for preventing or modulating thedevelopment of such diseases or disorders in an animal suspected ofbeing, or known to be, prone to such diseases or disorders.

[0022] In one embodiment, the invention provides methods of inhibitingthe expression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual, comprising the step of administering to said individual acompound of the invention targeted to a nucleic acid molecule encodingB7-1 or B7-2, wherein said compound specifically hybridizes with andinhibits the expression of a nucleic acid molecule encoding B7-1 orB7-2.

[0023] The invention further provides methods of inhibiting expressionof a nucleic acid molecule encoding B7-1 or B7-2 in an individual,comprising the step of administering to an individual a compound of theinvention which specifically hybridizes with at least an 8-nucleobaseportion of an active site on a nucleic acid molecule encoding B7-1 orB7-2. Regions in the nucleic acid which when hybridized to a compound ofthe invention effect significantly lower B7-1 or B7-2 expressioncompared to a control, are referred to as active sites.

[0024] The invention also provides methods of inhibiting expression of anucleic acid molecule encoding B7-1 or B7-2 in an individual, comprisingthe step of administering a compound of the invention targeted to anucleic acid molecule encoding B7-1 or B7-2, wherein the compoundspecifically hybridizes with the nucleic acid and inhibits expression ofB7-1 or B7-2.

[0025] In another aspect the invention provides methods of inhibitingexpression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual, comprising the step of administering a compound of theinvention targeted to a nucleic acid molecule encoding B7-1 or B7-2,wherein the compound specifically hybridizes with the nucleic acid andinhibits expression of B7-1 or B7-2, said compound comprising at least 8contiguous nucleobases of any one of the compounds of the invention.

[0026] The invention also provides methods of inhibiting the expressionof a nucleic acid molecule encoding B7-1 or B7-2 in an individual,comprising the step of administering a compound of the inventiontargeted to a nucleic acid molecule encoding B7-1 or B7-2, wherein thecompound specifically hybridizes with an active site in the nucleic acidand inhibits expression of B7-1 or B7-2, and the compound comprises atleast 8 contiguous nucleobases of any one of the compounds of theinvention.

[0027] In another aspect the invention provides methods of inhibitingexpression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual, comprising the step of administering an oligonucleotidemimetic compound targeted to a nucleic acid molecule encoding B7-1 orB7-2, wherein the compound specifically hybridizes with the nucleic acidand inhibits expression of B7-1 or B7-2, and the compound comprises atleast 8 contiguous nucleobases of a compound of the invention.

[0028] In another aspect, the invention provides methods of inhibitingthe expression of a nucleic acid molecule encoding B7-1 or B7-2 in anindividual comprising the step of administering a compound of theinvention target to a nucleic acid encoding B7-1 or B7-2, wherein thecompound inhibits B7-1 or B7-2 mRNA expression by at least 5% in 80%confluent HepG2 cells in culture at an optimum concentration compared toa control. In yet another aspect, the compounds inhibit expression ofmRNA encoding B7-1 or B7-2 in 80% confluent HepG2 cells in culture at anoptimum concentration by at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, or at least 50%,compared to a control.

[0029] The invention also relates to pharmaceutical compositions whichcomprise an antisense oligonucleotide to a B7 protein in combinationwith a second anti-inflammatory agent, such as a second antisenseoligonucleotide to a protein which mediates intercellular interactions,e.g., an intercellular adhesion molecule (ICAM) protein.

[0030] Methods comprising contacting animals with oligonucleotidesspecifically hybridizable with nucleic acids encoding B7 proteins areherein provided. These methods are useful as tools, for example, in thedetection and determination of the role of B7 protein expression invarious cell functions and physiological processes and conditions, andfor the diagnosis of conditions associated with such expression. Suchmethods can be used to detect the expression of 17 genes (i.e., B7-1 orB7-2) and are thus believed to be useful both therapeutically anddiagnostically. Methods of modulating the expression of B7 proteinscomprising contacting animals with oligonucleotides specificallyhybridizable with a B7 gene are herein provided. These methods arebelieved to be useful both therapeutically and diagnostically as aconsequence of the association between B7 expression and T cellactivation and proliferation. The present invention also comprisesmethods of inhibiting B7-associated activation of T cells using theoligonucleotides of the invention. Methods of treating conditions inwhich abnormal or excessive T cell activation and proliferation occursare also provided. These methods employ the oligonucleotides of theinvention and are believed to be useful both therapeutically and asclinical research and diagnostic tools. The oligonucleotides of thepresent invention may also be used for research purposes. Thus, thespecific hybridization exhibited by the oligonucleotides of the presentinvention may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

[0031] The methods disclosed herein are also useful, for example, asclinical research tools in the detection and determination of the roleof B7-1 or B7-2 expression in various immune system functions andphysiological processes and conditions, and for the diagnosis ofconditions associated with their expression. The specific hybridizationexhibited by the oligonucleotides of the present invention may be usedfor assays, purifications, cellular product preparations and in othermethodologies which may be appreciated by persons of ordinary skill inthe art. For example, because the oligonucleotides of this inventionspecifically hybridize to nucleic acids encoding B7 proteins, sandwichand other assays can easily be constructed to exploit this fact.Detection of specific hybridization of an oligonucleotide of theinvention with a nucleic acid encoding a B7 protein present in a samplecan routinely be accomplished. Such detection may include detectablylabeling an oligonucleotide of the invention by enzyme conjugation,radiolabeling or any other suitable detection system. A number of assaysmay be formulated employing the present invention, which assays willcommonly comprise contacting a tissue or cell sample with a detectablylabeled oligonucleotide of the present invention under conditionsselected to permit hybridization and measuring such hybridization bydetection of the label, as is appreciated by those of ordinary skill inthe art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a bar graph showing the inhibitory effect of theindicated oligonucleotides on B7-1 protein expression in COS-7 cells.

[0033]FIG. 2 is a dose-response curve showing the inhibitory effect ofoligonucleotides on cell surface expression of B7-1 protein. Solid line,ISIS 13812; dashed line, ISIS 13800; dotted line, ISIS 13805.

[0034]FIG. 3 is a bar graph showing the inhibitory effect of theindicated oligonucleotides on cell surface expression of B7-2 in COS-7cells.

[0035]FIG. 4 is a bar graph showing the inhibitory effect of theindicated oligonucleotides, including ISIS 10373 (a 20-mer) and ISIS10996 (a 15-mer) on cell surface expression of B7-2 in COS-7 cells.

[0036]FIG. 5 is a bar graph showing the specificity of inhibition ofB7-1 or B7-2 protein expression by oligonucleotides. Cross-hatched bars,B7-1 levels; striped bars, B7-2 levels.

[0037]FIG. 6 is a dose-response curve showing the inhibitory effect ofoligonucleotides having antisense sequences to ICAM-1 (ISIS 2302) orB7-2 (ISIS 10373) on cell surface expression of the ICAM-1 and B7-2proteins. Solid line with X's, levels of B7-1 protein on cells treatedwith ISIS 10373; dashed line with asterisks, levels of ICAM-1 protein oncells treated with ISIS 10373; solid line with triangles, levels of B7-1protein on cells treated with ISIS 2302; solid line with squares, levelsof ICAM-1 protein on cells treated with ISIS 10373.

[0038]FIG. 7 is a bar graph showing the effect of the indicatedoligonucleotides on T cell proliferation.

[0039]FIG. 8 is a dose-response curve showing the inhibitory effect ofoligonucleotides on murine B7-2 protein expression in COS-7 cells. Solidline with asterisks, ISIS 11696; dashed line with triangles, ISIS 11866.

[0040]FIG. 9 is a bar graph showing the effect of oligonucleotides ISIS11696 and ISIS 11866 on cell surface expression of murine B7-2 proteinin IC-21 cells. Left (black) bars, no oligonucleotide; middle bars, 3 μMindicated oligonucleotide; right bars, 10/i M indicated oligonucleotide.

[0041]FIG. 10 is a graph showing the effect of ISIS 17456 on severity ofEAE at various doses.

[0042] FIGS. 11A-B are graphs showing the detection of B7.2 mRNA (FIG.11A) and B7.1 mRNA (FIG. 11B) during the development ofovalbumin-induced asthma in a mouse model.

[0043]FIG. 12 is a graph showing that intratracheal administration ofISIS 121874, an antisense oligonucleotide targeted to mouse B7.2,following allergen challenge, reduces the airway response tomethacholine.

[0044]FIG. 13 is a graph showing the dose-dependent inhibition of thePenh response to 50 mg/ml methacholine by ISIS 121874. Penh is adimensionless parameter that is a function of total pulmonary airflow inmice (i.e., the sum of the airflow in the upper and lower respiratorytracts) during the respiratory cycle of the animal. The lower the PENH,the greater the airflow. The dose of ISIS 121874 is shown on the x-axis.

[0045]FIG. 14 is a graph showing the inhibition of allergen-inducedeosinophilia by ISIS 121874. The dose of ISIS 121874 is shown on thex-axis.

[0046]FIG. 15 is a graph showing the lung concentration-doserelationship for ISIS 121874 delivered by intratracheal administration.

[0047]FIG. 16 is a graph showing the retention of ISIS 121874 in lungtissue following single dose (0.3 mg/kg) intratracheal instillation inthe ovalbumin-induced mouse model of asthma.

[0048]FIG. 17 is a graph showing the effects of ISIS 121874, a 7 basepair mismatched control oligonucleotide (ISIS 131906) and a gap ablatedcontrol oligonucleotide which does not promote cleavage by RNase H (ISIS306058).

[0049] FIGS. 18A-B is a graph showing the effect of ISIS 121874 on B7.2(FIG. 18A) and B7.1 (FIG. 18B) mRNA in lung tissue ofovalbumin-challenged mice.

[0050] FIGS. 19A-B is a graph showing the effect of ISIS 121874 on B7.2(FIG. 19A) and B7.1 (FIG. 19B) mRNA in draining lymph nodes ofovalbumin-challenged mice.

[0051]FIG. 20 is a graph showing that treatment with an antisenseoligonucleotide targeted to B7.1 (ISIS 121844) reduces allergen-inducedeosinophilia in the ovalbumin-induced mouse model of asthma.

[0052] FIGS. 21A-B are graphs showing that treatment with ISIS 121844reduces the levels of B7.1 mRNA (FIG. 21A) and B7.2 mRNA (FIG. 21B) inthe mouse lung.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention employs oligonucleotides for use inantisense inhibition of the function of RNA and DNA encoding B7 proteinsincluding B7-1 and B7-2. The present invention also employsoligonucleotides which are designed to be specifically hybridizable toDNA or messenger RNA (mRNA) encoding such proteins and ultimately tomodulate the amount of such proteins transcribed from their respectivegenes. Such hybridization with mRNA interferes with the normal role ofmRNA and causes a modulation of its function in cells. The functions ofmRNA to be interfered with include all vital functions such astranslocation of the RNA to the site for protein translation, actualtranslation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and possibly even independent catalytic activitywhich may be engaged in by the RNA. The overall effect of suchinterference with mRNA function is modulation of the expression of a B7protein, wherein “modulation” means either an increase (stimulation) ora decrease (inhibition) in the expression of a B7 protein. In thecontext of the present invention, inhibition is the preferred form ofmodulation of gene expression.

[0054] Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described as“antisense.” Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents. For example,antisense oligonucleotides, which are able to inhibit gene expressionwith exquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes, for example to distinguishbetween the functions of various members of a biological pathway. Thisspecific inhibitory effect has, therefore, been harnessed by thoseskilled in the art for research uses.

[0055] “Hybridization”, in the context of this invention, means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases, usually on oppositenucleic acid strands or two regions of a nucleic acid strand. Guanineand cytosine are examples of complementary bases which are known to formthree hydrogen bonds between them. Adenine and thymine are examples ofcomplementary bases which form two hydrogen bonds between them.“Specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the DNA or RNA target and theoligonucleotide. It is understood that an oligonucleotide need not be100% complementary to its target nucleic acid sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when binding of the oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of activity, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment or, in the case of in vitro assays, underconditions in which the assays are conducted.

[0056] It is understood in the art that the sequence of the oligomericcompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. Moreover, an oligomeric compoundmay hybridize over one or more segments such that intervening oradjacent segments are not involved in the hybridization event (e.g., aloop structure or hairpin structure). It is preferred that theoligomeric compounds of the present invention comprise at least 70%sequence complementarity to a target region within the target nucleicacid, more preferably that they comprise 90% sequence complementarityand even more preferably comprise 95% sequence complementarity to thetarget region within the target nucleic acid sequence to which they aretargeted. For example, an oligomeric compound in which 18 of 20nucleobases of the oligomeric compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an oligomeric compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an oligomeric compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

[0057] In the present invention the phrase “stringent hybridizationconditions” or “stringent conditions” refers to conditions under whichan oligomeric compound of the invention will hybridize to its targetsequence, but to a minimal number of other sequences. Stringentconditions are sequence-dependent-and will vary with differentcircumstances and in the context of this invention; “stringentconditions” under which oligomeric compounds hybridize to a targetsequence are determined by the nature and composition of the oligomericcompounds and the assays in which they are being investigated.

[0058] The specificity and sensitivity of oligonucleotides is alsoharnessed by those of skill in the art for therapeutic uses. Forexample, the following U.S. patents demonstrate palliative, therapeuticand other methods utilizing antisense oligonucleotides. U.S. Pat. No.5,135,917 provides antisense oligonucleotides that inhibit humaninterleukin-1 receptor expression. U.S. Pat. No. 5,098,890 is directedto antisense oligonucleotides complementary to the c-myb oncogene andantisense oligonucleotide therapies for certain cancerous conditions.U.S. Pat. No. 5,087,617 provides methods for treating cancer patientswith antisense oligonucleotides. U.S. Pat. No. 5,166,195 providesoligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810 providesoligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA andinhibiting replication. U.S. Pat. No. 5,194,428 provides antisenseoligonucleotides having antiviral activity against influenza virus. U.S.Pat. No. 4,806,463 provides antisense oligonucleotides and methods usingthem to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717 providesoligonucleotides having a complementary base sequence to a portion of anoncogene. U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 aredirected to phosphorothioate oligonucleotide analogs used to preventreplication of foreign nucleic acids in cells. U.S. Pat. No. 4,689,320is directed to antisense oligonucleotides as antiviral agents specificto CMV. U.S. Pat. No. 5,098,890 provides oligonucleotides complementaryto at least a portion of the mRNA transcript of the human c-myb gene.U.S. Pat. No. 5,242,906 provides antisense oligonucleotides useful inthe treatment of latent EBV infections.

[0059] Oligonucleotides capable of modulating the expression of B7proteins represent a novel therapeutic class of anti-inflammatory agentswith activity towards a variety of inflammatory or autoimmune diseases,or disorders or diseases with an inflammatory component such as asthma,juvenile diabetes mellitus, myasthenia gravis, Graves' disease,rheumatoid arthritis, allograft rejection, inflammatory bowel disease,multiple sclerosis, psoriasis, lupus erythematosus, systemic lupuserythematosus, diabetes, multiple sclerosis, contact dermatitis, eczema,atopic dermatitis, seborrheic dermatitis, nummular dermatitis,generalized exfoliative dermatitis, rhinitis and various allergies. Inaddition, oligonucleotides capable of modulating the expression of B7proteins provide a novel means of manipulating the ex vivo proliferationof T cells.

[0060] It is preferred to target specific genes for antisense attack.“Targeting” an oligonucleotide to the associated nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a foreign nucleic acid from aninfectious agent. In the present invention, the target is a cellulargene associated with several immune system disorders and diseases (suchas inflammation and autoimmune diseases), as well as with ostensibly“normal” immune reactions (such as a host animal's rejection oftransplanted tissue), for which modulation is desired in certaininstances. The targeting process also includes determination of a region(or regions) within this gene for the oligonucleotide interaction tooccur such that the desired effect, either detection or modulation ofexpression of the protein, will result. Once the target region have beenidentified, oligonucleotides are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity to give the desired effect.

[0061] Generally, there are five regions of a gene that may be targetedfor antisense modulation: the 5′ untranslated region (hereinafter, the“5′-UTR”), the translation initiation codon region (hereinafter, the“tIR”), the open reading frame (hereinafter, the “ORF”), the translationtermination codon region (hereinafter, the “tTR”) and the 3′untranslated region (hereinafter, the “3“-UTR”). As is known in the art,these regions are arranged in a typical messenger RNA molecule in thefollowing order (left to right, 5′ to 3′): 5′-UTR, tIR, ORF, tTR,3′-UTR. As is known in the art, although some eukaryotic transcripts aredirectly translated, many ORFs contain one or more sequences, known as“introns,” which are excised from a transcript before it is translated;the expressed (unexcised) portions of the ORF are referred to as “exons”(Alberts et al., Molecular Biology of the Cell, 1983, Garland PublishingInc., New York, pp. 411-415). Furthermore, because many eukaryotic ORFsare a thousand nucleotides or more in length, it is often convenient tosubdivide the ORF into, e.g., the 5′ ORF region, the central ORF region,and the 3′ ORF region. In some instances, an ORF contains one or moresites that may be targeted due to some functional significance in vivo.Examples of the latter types of sites include intragenic stem-loopstructures (see, e.g., U.S. Pat. No. 5,512,438) and, in unprocessed mRNAmolecules, intron/exon splice sites. Within the context of the presentinvention, one preferred intragenic site is the region encompassing thetranslation initiation codon of the open reading frame (ORF) of thegene. Because, as is known in the art, the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon.” A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Furthermore, 5′-UUU functions as atranslation initiation codon in vitro (Brigstock et al., Growth Factors,1990, 4, 45; Gelbert et al., Somat. Cell. Mol. Genet., 1990, 16, 173;Gold and Stormo, in: Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology, Vol. 2, 1987, Neidhardt et al., eds.,American Society for Microbiology, Washington, D.C., p. 1303). Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine(prokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions,in order to generate related polypeptides having different aminoterminal sequences (Markussen et al., Development, 1995, 121, 3723; Gaoet al., Cancer Res., 1995, 55, 743; McDermott et al., Gene, 1992, 117,193; Perri et al., J. Biol. Chem., 1991, 266, 12536; French et al., J.Virol., 1989, 63, 3270; Pushpa-Rekha et al., J. Biol. Chem., 1995, 270,26993; Monaco et al., J. Biol. Chem., 1994, 269, 347; DeVirgilio et al.,Yeast, 1992, 8, 1043; Kanagasundaram et al., Biochim. Biophys. Acta,1992, 1171, 198; Olsen et al., Mol. Endocrinol., 1991, 5, 1246; Saul etal., Appl. Environ. Microbiol., 1990, 56, 3117; Yaoita et al., Proc.Natl. Acad. Sci. USA, 1990, 87, 7090; Rogers et al., EMBO J., 1990, 9,2273). In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding a B7 protein, regardless of the sequence(s) of such codons. Itis also known in the art that a translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively). The terms “start codon region” and “translationinitiation region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation termination codon.

[0062] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleicacid. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent intersugar(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced binding to target and increased stability in thepresence of nucleases.

[0063] While the preferred form of antisense compound is asingle-stranded antisense oligonucleotide, in many species theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

[0064] The first evidence that dsRNA could lead to gene silencing inanimals came in 1995 from work in the nematode, Caenorhabditis elegans(Guo and Kempheus, Cell, 1995, 81, 611-620).

[0065] Montgomery et al. have shown that the primary interferenceeffects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl.Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptionalantisense mechanism defined in Caenorhabditis elegans resulting fromexposure to double-stranded RNA (dsRNA) has since been designated RNAinterference (RNAi). This term has been generalized to meanantisense-mediated gene silencing involving the introduction of dsRNAleading to the sequence-specific reduction of endogenous targeted mRNAlevels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has beenshown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 2002, 295, 694-697).

[0066] Oligomer and Monomer Modifications

[0067] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside linkage or in conjunctionwith the sugar ring the backbone of the oligonucleotide. The normalinternucleoside linkage that makes up the backbone of RNA and DNA is a3′ to 5′ phosphodiester linkage.

[0068] Modified Internucleoside Linkages

[0069] Specific examples of preferred antisense oligomeric compoundsuseful in this invention include oligonucleotides containing modifiede.g. non-naturally occurring internucleoside linkages. As defined inthis specification, oligonucleotides having modified internucleosidelinkages include internucleoside linkages that retain a phosphorus atomand internucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

[0070] In the C. elegans system, modification of the internucleotidelinkage (phosphorothioate) did not significantly interfere with RNAiactivity. Based on this observation, it is suggested that certainpreferred oligomeric compounds of the invention can also have one ormore modified internucleoside linkages. A preferred phosphoruscontaining modified internucleoside linkage is the phosphorothioateinternucleoside linkage.

[0071] Preferred modified oligonucleotide backbones containing aphosphorus atom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0072] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0073] In more preferred embodiments of the invention, oligomericcompounds have one or more phosphorothioate and/or heteroatominternucleoside linkages, in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃) —N(CH₃) —CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester internucleotide linkage isrepresented as —O—P(═O)(OH)—O—CH₂—]. The MMI type internucleosidelinkages are disclosed in the above referenced U.S. Pat. No. 5,489,677.Preferred amide internucleoside linkages are disclosed in the abovereferenced U.S. Pat. No. 5,602,240.

[0074] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0075] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0076] Oligomer Mimetics

[0077] Another preferred group of oligomeric compounds amenable to thepresent invention includes oligonucleotide mimetics. The term mimetic asit is applied to oligonucleotides is intended to include oligomericcompounds wherein only the furanose ring or both the furanose ring andthe internucleotide linkage are replaced with novel groups, replacementof only the furanose ring is also referred to in the art as being asugar surrogate. The heterocyclic base moiety or a modified heterocyclicbase moiety is maintained for hybridization with an appropriate targetnucleic acid. One such oligomeric compound, an oligonucleotide mimeticthat has been shown to have excellent hybridization properties, isreferred to as a peptide nucleic acid (PNA). In PNA oligomericcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The nucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited. States patents that teach the preparation of PNA oligomericcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA oligomeric compounds can be found inNielsen et al., Science, 1991, 254, 1497-1500.

[0078] One oligonucleotide mimetic that has been reported to haveexcellent hybridization properties is peptide nucleic acids (PNA). Thebackbone in PNA compounds is two or more linked aminoethylglycine unitswhich gives PNA an amide containing backbone. The heterocyclic basemoieties are bound directly or indirectly to aza nitrogen atoms of theamide portion of the backbone. Representative United States patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which isherein incorporated by reference. Further teaching of PNA compounds canbe found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0079] PNA has been modified to incorporate numerous modifications sincethe basic PNA structure was first prepared. The basic structure is shownbelow:

[0080] wherein

[0081] Bx is a heterocyclic base moiety;

[0082] T₄ is hydrogen, an amino protecting group, —C(O)R₅, substitutedor unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

[0083] T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via theα-amino group or optionally through the β-amino group when the aminoacid is lysine or ornithine or a peptide derived from D, L or mixed Dand L amino acids linked through an amino group, a chemical functionalgroup, a reporter group or a conjugate group;

[0084] Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

[0085] Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the o-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

[0086] Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl,—C(═O)—CH₃, benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

[0087] each J is O, S or NH;

[0088] R₅ is a carbonyl protecting group; and

[0089] n is from 2 to about 50.

[0090] Another class of oligonucleotide mimetic that has been studied isbased on linked morpholino units (morpholino nucleic acid) havingheterocyclic bases attached to the morpholino ring. A number of linkinggroups have been reported that link the morpholino monomeric units in amorpholino nucleic acid. A preferred class of linking groups have beenselected to give a non-ionic oligomeric compound. The non-ionicmorpholino-based oligomeric compounds are less likely to have undesiredinteractions with cellular proteins. Morpholino-based oligomericcompounds are non-ionic mimics of oligonucleotides which are less likelyto form undesired interactions with cellular proteins (Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510).Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No.5,034,506, issued Jul. 23, 1991. The morpholino class of oligomericcompounds have been prepared having a variety of different linkinggroups joining the monomeric subunits.

[0091] Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

[0092] wherein

[0093] T₁ is hydroxyl or a protected hydroxyl;

[0094] T₅ is hydrogen or a phosphate or phosphate derivative;

[0095] L₂ is a linking group; and

[0096] n is from 2 to about 50.

[0097] A further class of oligonucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present inan DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Ingeneral the incorporation of CeNA monomers into a DNA chain increasesits stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes. The study of incorporating CeNA structures into naturalnucleic acid structures was shown by NMR and circular dichroism toproceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. Coli RNase resulting in cleavage of the targetRNA strand.

[0098] The general formula of CeNA is shown below:

[0099] wherein

[0100] each Bx is a heterocyclic base moiety;

[0101] T₁ is hydroxyl or a protected hydroxyl; and

[0102] T₂ is hydroxyl or a protected hydroxyl.

[0103] Another class of oligonucleotide mimetic (anhydrohexitol nucleicacid) can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

[0104] A further preferred modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom ofthe sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage therebyforming a bicyclic sugar moiety. The linkage is preferably a methylene(—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atomwherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNAand LNA analogs display very high duplex thermal stabilities withcomplementary DNA and RNA (Tm=+3 to +10 C), stability towards3′-exonucleolytic degradation and good solubility properties. The basicstructure of LNA showing the bicyclic ring system is shown below:

[0105] The conformations of LNAs determined by 2D NMR spectroscopy haveshown that the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

[0106] LNA has been shown to form exceedingly stable LNA:LNA duplexes(Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

[0107] LNAs also form duplexes with complementary DNA, RNA or LNA withhigh thermal affinities. Circular dichroism (CD) spectra show thatduplexes involving fully modified LNA (esp. LNA:RNA) structurallyresemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR)examination of an LNA:DNA duplex confirmed the 3′-endo conformation ofan LNA monomer. Recognition of double-stranded DNA has also beendemonstrated suggesting strand invasion by LNA. Studies of mismatchedsequences show that LNAs obey the Watson-Crick base pairing rules withgenerally improved selectivity compared to the corresponding unmodifiedreference strands.

[0108] Novel types of LNA-oligomeric compounds, as well as the LNAs, areuseful in a wide range of diagnostic and therapeutic applications. Amongthese are antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs. Potent and nontoxic antisense oligonucleotidescontaining LNAs have been described (Wahlestedt et al., Proc. Natl.Acad. Sci. U.S.A., 2000, 97, 5633-5638.) The authors have demonstratedthat LNAs confer several desired properties to antisense agents. LNA/DNAcopolymers were not degraded readily in blood serum and cell extracts.LNA/DNA copolymers exhibited potent antisense activity in assay systemsas disparate as G-protein-coupled receptor signaling in living rat brainand detection of reporter genes in Escherichia coli. Lipofectin-mediatedefficient delivery of LNA into living human breast cancer cells has alsobeen accomplished.

[0109] The synthesis and preparation of the LNA monomers adenine,cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along withtheir oligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

[0110] The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs,have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998,8, 2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2‘-methylamino-LNA’s have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

[0111] Further oligonucleotide mimetics have been prepared to includebicyclic and tricyclic nucleoside analogs having the formulas (amiditemonomers shown):

[0112] (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439;Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberget al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These modifiednucleoside analogs have been oligomerized using the phosphoramiditeapproach and the resulting oligomeric compounds containing tricyclicnucleoside analogs have shown increased thermal stabilities (Tm's) whenhybridized to DNA, RNA and itself. Oligomeric compounds containingbicyclic nucleoside analogs have shown thermal stabilities approachingthat of DNA duplexes.

[0113] Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids incorporate a phosphorus group in abackbone the backbone. This class of olignucleotide mimetic is reportedto have useful physical and biological and pharmacological properties inthe areas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

[0114] The general formula (for definitions of Markush variables see:U.S. Pat. Nos. 5,874,553 and 6,127,346 herein incorporated by referencein their entirety) is shown below.

[0115] Another oligonucleotide mimetic has been reported wherein thefuranosyl ring has been replaced by a cyclobutyl moiety.

[0116] Modified Sugars

[0117] Oligomeric compounds of the invention may also contain one ormore substituted sugar moieties. Preferred oligomeric compounds comprisea sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-,S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃] ₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise a sugarsubstituent group selected from: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃) 2

[0118] Other preferred sugar substituent groups include methoxy(—O—CH₃), aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligomeric compound, particularly the 3′position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0119] Further representative sugar substituent groups include groups offormula I_(a) or II_(a):

[0120] wherein:

[0121] R_(b) is O, S or NH;

[0122] R_(d) is a single bond, O, S or C(═O);

[0123] R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)), N═C(R_(p)) (R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);

[0124] R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

[0125] R_(r) is —R_(x)—R_(y);

[0126] each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or aconjugate group, wherein the substituent groups are selected fromhydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

[0127] or optionally, R_(u) and R_(v), together form a phthalimidomoiety with the nitrogen atom to which they are attached;

[0128] each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy,allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy,2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

[0129] R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

[0130] R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

[0131] R_(x) is a bond or a linking moiety;

[0132] R_(y) is a chemical functional group, a conjugate group or asolid support medium;

[0133] each R_(m) and R_(n) is, independently, H, a nitrogen protectinggroup, substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, wherein the substituent groups are selected from hydroxyl,amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺, N(R_(u)) (R_(v)),guanidino and acyl where said acyl is an acid amide or an ester;

[0134] or R_(m) and R_(n), together, are a nitrogen protecting group,are joined in a ring structure that optionally includes an additionalheteroatom selected from N and O or are a chemical functional group;

[0135] R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

[0136] each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

[0137] R_(f), R_(g) and R_(h) comprise a ring system having from about 4to about 7 carbon atoms or having from about 3 to about 6 carbon atomsand 1 or 2 heteroatoms wherein said heteroatoms are selected fromoxygen, nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

[0138] R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms,N(R_(k))(R_(m))OR_(k), halo, SR_(k) or CN;

[0139] m_(a) is 1 to about 10;

[0140] each m_(b) is, independently, 0 or 1;

[0141] m_(c) is 0 or an integer from 1 to 10;

[0142] m_(d) is an integer from 1 to 10;

[0143] m_(e) is from 0, 1 or 2; and

[0144] provided that when m_(c) is 0, m_(d) is greater than 1.

[0145] Representative substituents groups of Formula I are disclosed inU.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998,entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated byreference in its entirety.

[0146] Representative cyclic substituent groups of Formula II aredisclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27,1998, entitled “RNA Targeted 2′-Oligomeric compounds that areConformationally Preorganized,” hereby incorporated by reference in itsentirety.

[0147] Particularly preferred sugar substituent groups includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, (CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10.

[0148] Representative guanidino substituent groups that are shown informula III and IV are disclosed in co-owned U.S. patent applicationSer. No. 09/349,040, entitled “Functionalized Oligomers”, filed Jul. 7,1999, hereby incorporated by reference in its entirety.

[0149] Representative acetamido substituent groups are disclosed in U.S.Pat. No. 6,147,200 which is hereby incorporated by reference in itsentirety.

[0150] Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

[0151] Modified Nucleobases/Naturally Occurring Nucleobases

[0152] Oligomeric compounds may also include nucleobase (often referredto in the art simply as “base” or “heterocyclic base moiety”)modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases also referred herein as heterocyclic base moietiesinclude other synthetic and natural nucleobases such as5-methylcytosine-(5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

[0153] Heterocyclic base moieties may also include those in which thepurine or pyrimidine base is replaced with other heterocycles, forexample 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research: and Applications, pages289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0154] In one aspect of the present invention oligomeric compounds areprepared having polycyclic heterocyclic compounds in place of one ormore heterocyclic base moieties. A number of tricyclic heterocycliccompounds have been previously reported. These compounds are routinelyused in antisense applications to increase the binding properties of themodified strand to a target strand. The most studied modifications aretargeted to guanosines hence they have been termed G-clamps or cytidineanalogs. Many of these polycyclic heterocyclic compounds have thegeneral formula:

[0155] Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀₌O,R₁₁-R₁₄=H) [Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16,1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀=S, R₁₁-R₁₄=H), [Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀=O,R₁₁-R₁₄=F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.Patent Application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. Patent Application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are commonly owned with this applicationand are herein incorporated by reference in their entirety).

[0156] Further helix-stabilizing properties have been observed when acytosine analog/substitute has an aminoethoxy moiety attached to therigid 1,3-diazaphenoxazine-2-one scaffold (R₁₀₌O, R₁₁=—O—(CH₂)₂—NH₂,R₁₂₋₁₄=H) [Lin, K.-Y., Matteucci, M. J. Am. Chem. Soc. 1998, 120,8531-8532]. Binding studies demonstrated that a single incorporationcould enhance the binding affinity of a model oligonucleotide to itscomplementary target DNA or RNA with a ΔT_(m) of up to 180 relative to5-methyl cytosine (dC5^(me)), which is the highest known affinityenhancement for a single modification, yet. On the other hand, the gainin helical stability does not compromise the specificity of theoligonucleotides. The T_(m) data indicate an even greater discriminationbetween the perfect match and mismatched sequences compared to dC5^(me).It was suggested that the tethered amino group serves as an additionalhydrogen bond donor to interact with the Hoogsteen face, namely the O6,of a complementary guanine thereby forming 4 hydrogen bonds. This meansthat the increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

[0157] Further tricyclic heterocyclic compounds and methods of usingthem that are amenable to the present invention are disclosed in U.S.Pat. No. 6,028,183, which issued on May 22, 2000, and U.S. Pat. No.6,007,992, which issued on Dec. 28, 1999, the contents of both arecommonly assigned with this application and are incorporated herein intheir entirety.

[0158] The enhanced binding affinity of the phenoxazine derivativestogether with their uncompromised sequence specificity makes themvaluable nucleobase analogs for the development of more potentantisense-based drugs. In fact, promising data have been derived from invitro experiments demonstrating that heptanucleotides containingphenoxazine substitutions are capable to activate RNaseH, enhancecellular uptake and exhibit an increased antisense activity [Lin, K-Y;Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532]. The activityenhancement was even more pronounced in case of G-clamp, as a singlesubstitution was shown to significantly improve the in vitro potency ofa 20mer 2′-deoxyphosphorothioate oligonucleotides [Flanagan, W.-M.;Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci,M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, tooptimize oligonucleotide design and to better understand the impact ofthese heterocyclic modifications on the biological activity, it isimportant to evaluate their effect on the nuclease stability of theoligomers.

[0159] Further modified polycyclic heterocyclic compounds useful asheterocyclcic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patentapplication Ser. No. 09/996,292 filed Nov. 28, 2001, certain of whichare commonly owned with the instant application, and each of which isherein incorporated by reference.

[0160] The oligonucleotides of the present invention also includevariants in which a different base is present at one or more of thenucleotide positions in the oligonucleotide. For example, if the firstnucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the oligonucleotide. Thus, a 20-mer may comprise60 variations (20 positions×3 alternates at each position) in which theoriginal nucleotide is substituted with any of the three alternatenucleotides. These oligonucleotides are then tested using the methodsdescribed herein to determine their ability to inhibit expression of HCVmRNA and/or HCV replication.

[0161] Conjugates

[0162] A further preferred substitution that can be appended to theoligomeric compounds of the invention involves the linkage of one ormore moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the resulting oligomeric compounds.In one embodiment such modified oligomeric compounds are prepared bycovalently attaching conjugate groups to functional groups such ashydroxyl or amino groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugates groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve oligomer uptake, enhance oligomerresistance to degradation, and/or strengthen sequence-specifichybridization with RNA. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove oligomer uptake, distribution, metabolism or excretion.Representative conjugate groups are disclosed in International PatentApplication PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure ofwhich is incorporated herein by reference. Conjugate moieties includebut are not limited to lipid moieties such as a cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-o-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

[0163] The oligomeric compounds of the invention may also be conjugatedto active drug substances, for example, aspirin, warfarin,phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0164] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0165] Chimeric Oligomeric Compounds

[0166] It is not necessary for all positions in an oligomeric compoundto be uniformly modified, and in fact more than one of theaforementioned modifications may be incorporated in a single oligomericcompound or even at a single monomeric subunit such as a nucleosidewithin a oligomeric compound. The present invention also includesoligomeric compounds which are chimeric oligomeric compounds. “Chimeric”oligomeric compounds or “chimeras,” in the context of this invention,are oligomeric compounds that contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

[0167] Chimeric oligomeric compounds typically contain at least oneregion modified so as to confer increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. An additional region of theoligomeric compound may serve as a substrate for enzymes capable ofcleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is acellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligomeric compounds when chimeras are used, compared to forexample phosphorothioate deoxyoligonucleotides hybridizing to the sametarget region. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0168] Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Such oligomeric compounds have also been referred to in the artas hybrids hemimers, gapmers or inverted gapmers. Representative UnitedStates patents that teach the preparation of such hybrid structuresinclude, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797;5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

[0169] 3′-Endo Modifications

[0170] In one aspect of the present invention oligomeric compoundsinclude nucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2α-deoxy-2′-F-nucleosides appears efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but aren'tlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric triggers of RNAi having one or morenucleosides modified in such a way as to favor a C3′-endo typeconformation.

[0171] Nucleoside conformation is influenced by various factorsincluding substitution at the 2′, 3′ or 4′-positions of thepentofuranosyl sugar. Electronegative substituents generally prefer theaxial positions, while sterically demanding substituents generallyprefer the equatorial positions (Principles of Nucleic Acid Structure,Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ positionto favor the 3′′-endo conformation can be achieved while maintaining the2′-OH as a recognition element, as illustrated in FIG. 2, below (Galloet al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org.Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999),64, 747-754.) Alternatively, preference for the 3′-endo conformation canbe achieved by deletion of the 2′-OH as exemplified by 2′deoxy-2°F.-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841),which adopts the 3′-endo conformation positioning the electronegativefluorine atom in the axial position. Other modifications of the ribosering, for example substitution at the 4′-position to give 4′-F modifiednucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters(1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41,3010-3017), or for example modification to yield methanocarba nucleosideanalogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 andLee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11,1333-1337) also induce preference for the 3′-endo conformation. Alongsimilar lines, oligomeric triggers of RNAi response might be composed ofone or more nucleosides modified in such a way that conformation islocked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA,Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridgedNucleic Acids (ENA, Morita et al, Bioorganic & Medicinal ChemistryLetters (2002), 12, 73-76.) Examples of modified nucleosides amenable tothe present invention are shown below in Table I. These examples aremeant to be representative and not exhaustive. TABLE I

[0172] The preferred conformation of modified nucleosides and theiroligomers can be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligoncleotides of the presentinvention. The synthesis of numerous of the modified nucleosidesamenable to the present invention are known in the art (see for example,Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend,1988, Plenum press., and the examples section below.) Nucleosides knownto be inhibitors/substrates for-RNA dependent RNA polymerases (forexample HCV NS5B).

[0173] In one aspect, the present invention is directed tooligonucleotides that are prepared having enhanced properties comparedto native RNA against nucleic acid targets. A target is identified andan oligonucleotide is selected having an effective length and sequencethat is complementary to a portion of the target sequence. Eachnucleoside of the selected sequence is scrutinized for possibleenhancing modifications. A preferred modification would be thereplacement of one or more RNA nucleosides with nucleosides that havethe same 3′-endo conformational geometry. Such modifications can enhancechemical and nuclease stability relative to native RNA while at the sametime being much cheaper and easier to synthesize and/or incorporate intoan oligonulceotide. The selected sequence can be further divided intoregions and the nucleosides of each region evaluated for enhancingmodifications that can be the result of a chimeric configuration.Consideration is also given to the 5′ and 3′-termini as there are oftenadvantageous modifications that can be made to one or more of theterminal nucleosides. The oligomeric compounds of the present inventioninclude at least one 5′-modified phosphate group on a single strand oron at least one 5′-position of a double stranded sequence or sequences.Further modifications are also considered such as internucleosidelinkages, conjugate groups, substitute sugars or bases, substitution ofone or more nucleosides with nucleoside mimetics and any othermodification that can enhance the selected sequence for its intendedtarget. The terms used to describe the conformational geometry ofhomoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. Therespective conformational geometry for RNA and DNA duplexes wasdetermined from X-ray diffraction analysis of nucleic acid fibers(Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) Ingeneral, RNA:RNA duplexes are more stable and have higher meltingtemperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles ofNucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik etal., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic AcidsRes., 1997, 25, 2627-2634). The increased stability of RNA has beenattributed to several structural features, most notably the improvedbase stacking interactions that result from an A-form geometry (Searleet al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., alsodesignated as Northern pucker, which causes the duplex to favor theA-form geometry. In addition, the 2′ hydroxyl groups of RNA can form anetwork of water mediated hydrogen bonds that help stabilize the RNAduplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the otherhand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., alsoknown as Southern pucker, which is thought to impart a less stableB-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and 04′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a 04′-endo pucker contribution.

[0174] DNA:RNA hybrid duplexes, however, are usually less stable thanpure RNA:RNA duplexes, and depending on their sequence may be eithermore or less stable than DNA:DNA duplexes (Searle et al., Nucleic AcidsRes., 1993, 21, 2051-2056). The structure of a hybrid duplex isintermediate between A- and B-form geometries, which may result in poorstacking interactions (Lane et al., Eur. J. Biochem., 1993, 21)5,297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez etal., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol.,1996, 264, 521-533). The stability of the duplex formed between a targetRNA and a synthetic sequence is central to therapies such as but notlimited to antisense and RNA interference as these mechanisms requirethe binding of a synthetic oligonucleotide strand to an RNA targetstrand. In the case of antisense, effective inhibition of the mRNArequires that the antisense DNA have a very high binding affinity withthe mRNA. Otherwise the desired interaction between the syntheticoligonucleotide strand and target mRNA strand will occur infrequently,resulting in decreased efficacy.

[0175] One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

[0176] As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

[0177] One synthetic 2′-modification that imparts increased nucleaseresistance and a very high binding affinity to nucleotides is the2-methoxyethoxy (2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J.Biol. Chem., 1997, 272, 11944-12000). One of the immediate advantages ofthe 2′-MOE substitution is the improvement in binding affinity, which isgreater than many similar 2′ modifications such as O-methyl, O-propyl,and O-aminopropyl. Oligonucleotides having the 2′-O-methoxyethylsubstituent also have been shown to be antisense inhibitors of geneexpression with promising features for in vivo use (Martin, P., Helv.Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50,168-7176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; andAltmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative toDNA, the oligonucleotides having the 2′-MOE modification displayedimproved RNA affinity and higher nuclease resistance. Chimericoligonucleotides having 2′-MOE substituents in the wing nucleosides andan internal region of deoxy-phosphorothioate nucleotides (also termed agapped oligonucleotide or gapmer) have shown effective reduction in thegrowth of tumors in animal models at low doses. 2′-MOE substitutedoligonucleotides have also shown outstanding promise as antisense agentsin several disease states. One such MOE substituted oligonucleotide ispresently being investigated in clinical trials for the treatment of CMVretinitis.

[0178] Chemistries Defined

[0179] Unless otherwise defined herein, alkyl means C₁-C₁₂, preferablyC₁-C₈, and more preferably C₁-C₆, straight or (where possible) branchedchain aliphatic hydrocarbyl.

[0180] Unless otherwise defined herein, heteroalkyl means C₁-C₁₂,preferably C₁-C₈, and more preferably C₁-C₆, straight or (wherepossible) branched chain aliphatic hydrocarbyl containing at least one,and preferably about 1 to about 3, hetero atoms in the chain, includingthe terminal portion of the chain. Preferred heteroatoms include N, Oand S.

[0181] Unless otherwise defined herein, cycloalkyl means C₃-C₁₂,preferably C₃-C₈, and more preferably C₃-C₆, aliphatic hydrocarbyl ring.

[0182] Unless otherwise defined herein, alkenyl means C₂-C₁₂, preferablyC₂-C₈, and more preferably C₂-C₆ alkenyl, which may be straight or(where possible) branched hydrocarbyl moiety, which contains at leastone carbon-carbon double bond.

[0183] Unless otherwise defined herein, alkynyl means C₂-C₁₂, preferablyC₂-C₈, and more preferably C₂-C₆ alkynyl, which may be straight or(where possible) branched hydrocarbyl moiety, which contains at leastone carbon-carbon triple bond.

[0184] Unless otherwise defined herein, heterocycloalkyl means a ringmoiety containing at least three ring members, at least one of which iscarbon, and of which 1, 2 or three ring members are other than carbon.Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred heterocycloalkyl groupsinclude morpholino, thiomorpholino, piperidinyl, piperazinyl,homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino,pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl,tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl,furanyl, pyranyl, and tetrahydroisothiazolyl.

[0185] Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Preferred aryl rings haveabout 6 to about 20 ring carbons. Especially preferred aryl ringsinclude phenyl, napthyl, anthracenyl, and phenanthrenyl.

[0186] Unless otherwise defined herein, hetaryl means a ring moietycontaining at least one fully unsaturated ring, the ring consisting ofcarbon and non-carbon atoms. Preferably the ring system contains about 1to about 4 rings. Preferably the number of carbon atoms varies from 1 toabout 12, preferably 1 to about 6, and the total number of ring membersvaries from three to about 15, preferably from about 3 to about 8.Preferred ring heteroatoms are N, O and S. Preferred hetaryl moietiesinclude pyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl,pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl,benzothiophenyl, etc.

[0187] Unless otherwise defined herein, where a moiety is defined as acompound moiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryland alkyl), etc., each of the sub-moieties is as defined herein.

[0188] Unless otherwise defined herein, an electron withdrawing group isa group, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

[0189] Unless otherwise defined herein, the terms halogen and halo havetheir ordinary meanings. Preferred halo (halogen) substituents are Cl,Br, and I.

[0190] The aforementioned optional substituents are, unless otherwiseherein defined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc.

[0191] In all the preceding formulae, the squiggle (˜) indicates a bondto an oxygen or sulfur of the 5′-phosphate.

[0192] Phosphate protecting groups include those described in U.S. Pat.No. US 5,760,209, US 5,614,621, US 6,051,699, US 6,020,475, US6,326,478, US 6,169,177, US 6,121,437, US 6,465,628 each of which isexpressly incorporated herein by reference in its entirety.

[0193] The oligonucleotides in accordance with this invention (singlestranded or double stranded) preferably comprise from about 8 to about80 nucleotides, more preferably from about 12-50 nucleotides and mostpreferably from about 15 to 30 nucleotides. As is known in the art, anucleotide is a base-sugar combination suitably bound to an adjacentnucleotide through a phosphodiester, phosphorothioate or other covalentlinkage.

[0194] The oligonucleotides of the present invention also includevariants in which a different base is present at one or more of thenucleotide positions in the oligonucleotide. For example, if the firstnucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the oligonucleotide. Thus, a 20-mer may comprise60 variations (20 positions×3 alternates at each position) in which theoriginal nucleotide is substituted with any of the three alternatenucleotides. These oligonucleotides are then tested using the methodsdescribed herein to determine their ability to inhibit expression ofB7.1 or B7.2 mRNA.

[0195] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by-severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0196] The oligonucleotides of the present invention can be utilized astherapeutic compounds, diagnostic tools and as research reagents andkits. The term “therapeutic uses” is intended to encompass prophylactic,palliative and curative uses wherein the oligonucleotides of theinvention are contacted with animal cells either in vivo or ex vivo.When contacted with animal cells ex vivo, a therapeutic use includesincorporating such cells into an animal after treatment with one or moreoligonucleotides of the invention. While not intending to be bound to aparticular utility, the ex vivo modulation of, e.g., T cellproliferation by the oligonucleotides of the invention can be employedin, for example, potential therapeutic modalities wherein it is desiredto modulate the expression of a B7 protein in APCs.

[0197] As an example, oligonucleotides that inhibit the expression ofB7-1 proteins are expected to enhance the availability of B7-2 proteinson the surface of APCs, thus increasing the costimulatory effect of B7-2on T cells ex vivo (Levine et al., Science, 1996, 272, 1939).

[0198] For therapeutic uses, an animal suspected of having a disease ordisorder which can be treated or prevented by modulating the expressionor activity of a B7 protein is, for example, treated by administeringoligonucleotides in accordance with this invention. The oligonucleotidesof the invention can be utilized in pharmaceutical compositions byadding an effective amount of an oligonucleotide to a suitablepharmaceutically acceptable diluent or carrier. Workers in the fieldhave identified antisense, triplex and other oligonucleotidecompositions which are capable of modulating expression of genesimplicated in viral, fungal and metabolic diseases. Antisenseoligonucleotides have been safely administered to humans and severalclinical trials are presently underway. It is thus established thatoligonucleotides can be useful therapeutic instrumentalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

[0199] The oligonucleotides of the present invention can be further usedto detect the presence of B7-specific nucleic acids in a cell or tissuesample. For example, radiolabeled oligonucleotides can be prepared by³²P labeling at the 5′ end with polynucleotide kinase (Sambrook et al.,Molecular Cloning. A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, Volume 2, pg. 10.59). Radiolabeled oligonucleotides arethen contacted with cell or tissue samples suspected of containing B7message RNAs (and thus B7 proteins), and the samples are washed toremove unbound oligonucleotide. Radioactivity remaining in the sampleindicates the presence of bound oligonucleotide, which in turn indicatesthe presence of nucleic acids complementary to the oligonucleotide, andcan be quantitated using a scintillation counter or other routine means.Expression of nucleic acids encoding these proteins is thus detected.

[0200] Radiolabeled oligonucleotides of the present invention can alsobe used to perform autoradiography of tissues to determine thelocalization, distribution and quantitation of B7 proteins for research,diagnostic or therapeutic purposes. In such studies, tissue sectionsare; treated with radiolabeled oligonucleotide and washed as describedabove, then exposed to photographic emulsion according to routineautoradiography procedures. The emulsion, when developed, yields animage of silver grains over the regions expressing a B7 gene.Quantitation of the silver grains permits detection of the expression ofmRNA molecules encoding these proteins and permits targeting ofoligonucleotides to these areas.

[0201] Analogous assays for fluorescent detection of expression of B7nucleic acids can be developed using oligonucleotides of the presentinvention which are conjugated with fluorescein or other fluorescenttags instead of radiolabeling. Such conjugations are routinelyaccomplished during solid phase synthesis using fluorescently-labeledamidites or controlled pore glass (CPG) columns. Fluorescein-labeledamidites and CPG are available from, e.g., Glen Research, Sterling Va.

[0202] The present invention employs oligonucleotides targeted tonucleic acids encoding B7 proteins and oligonucleotides targeted tonucleic acids encoding such proteins. Kits for detecting the presence orabsence of expression of a B7 protein may also be prepared. Such kitsinclude an oligonucleotide targeted to an appropriate gene, i.e., a geneencoding a B7 protein. Appropriate kit and assay formats, such as, e.g.,“sandwich” assays, are known in the art and can easily be adapted foruse with the oligonucleotides of the invention. Hybridization of theoligonucleotides of the invention with a nucleic acid encoding a B7protein can be detected by means known in the art. Such means mayinclude conjugation of an enzyme to the oligonucleotide, radiolabellingof the oligonucleotide or any other suitable detection systems. Kits fordetecting the presence or absence of a B7 protein may also be prepared.

[0203] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleotides. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. “Complementary,” as used herein, refers tothe capacity for precise pairing between two nucleotides. For example,if a nucleotide at a certain position of an oligonucleotide is capableof hydrogen bonding with a nucleotide at the same position of a DNA orRNA molecule, then the oligonucleotide and the DNA or RNA are consideredto be complementary to each other at that position. The oligonucleotideand the DNA or RNA are complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. It is understood in the art that an oligonucleotide need notbe 100% complementary to its target DNA sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA to cause adecrease or loss of function, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed.

[0204] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered an oligonucleotide in accordance with the invention,commonly in a pharmaceutically acceptable carrier, in doses ranging from0.01 μg to 100 g per kg of body weight depending on the age of thepatient and the severity of the disorder or disease state being treated.Further, the treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease or disorder,its severity and the overall condition of the patient, and may extendfrom once daily to once every 20 years. Following treatment, the patientis monitored for changes in his/her condition and for alleviation of thesymptoms of the disorder or disease state. The dosage of theoligonucleotide may either be increased in the event the patient doesnot respond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disorder or diseasestate is observed, or if the disorder or disease state has been ablated.

[0205] In some cases, it may be more effective to treat a patient withan oligonucleotide of the invention in conjunction with othertherapeutic modalities in order to increase the efficacy of a treatmentregimen. In the context of the invention, the term “treatment regimen”is meant to encompass therapeutic, palliative and prophylacticmodalities. In a preferred embodiment, the oligonucleotides of theinvention are used in conjunction with an anti-inflammatory and/orimmunosuppressive agent, preferably one or more antisenseoligonucleotides targeted to an intercellular adhesion molecule (ICAM),preferably to ICAM-1. Other anti-inflammatory and/or immunosuppressiveagents that may be used in combination with the oligonucleotides of theinvention include, but are not limited to, soluble ICAM proteins (e.g.,sICAM-1), antibody-toxin conjugates, prednisone, methylprednisolone,azathioprine, cyclophosphamide, cyclosporine, interferons,sympathomimetics, conventional antihistamines (histamine H₁ receptorantagonists, including, for example, brompheniramine maleate,chlorpheniramine maleate, dexchlorpheniramine maleate, tripolidine HCl,carbinoxamine maleate, clemastine fumarate, dimenhydrinate,diphenhydramine HCl, diphenylpyraline HCl, doxylamine succinate,tripelennamine citrate, tripelennamine HCl, cyclizine HCl, hydroxyzineHCl, meclizine HCl, methdilazine HCl, promethazine HCl, trimeprazinetartrate, azatadine maleate, cyproheptadine HCl, terfenadine, etc.),histamine H₂ receptor antagonists (e.g., ranitidine). See, generally,The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,eds., 1987, Rahway, N.J., pages 302-336 and 2516-2522). When used withthe compounds of the invention, such agents may be used individually,sequentially, or in combination with one or more other such agents.

[0206] In another preferred embodiment of the invention, an antisenseoligonucleotide targeted to one B7 mRNA species (e.g., B7-1) is used incombination with an antisense oligonucleotide targeted to a second B7mRNA species (e.g., B7-2) in order to inhibit the costimulatory effectof B7 molecules to a more extensive degree than can be achieved witheither oligonucleotide used individually. In a related version of thisembodiment, two or more oligonucleotides of the invention, each targetedto an alternatively spliced B7-1 or B7-2 mRNA, are combined with eachother in order to inhibit expression of both forms of the alternativelyspliced mRNAs. It is known in the art that, depending on the specificityof the modulating agent employed, inhibition of one form of analternatively spliced mRNA may not result in a sufficient reduction ofexpression for a given condition to be manifest. Thus, such combinationsmay, in some instances, be desired to inhibit the expression of aparticular B7 gene to an extent necessary to practice one of the methodsof the invention. Following successful treatment, it may be desirable tohave the patient undergo maintenance therapy to prevent the recurrenceof the disease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years. In the case of in individualknown or suspected of being prone to an autoimmune or inflammatorycondition, prophylactic effects may be achieved by administration ofpreventative doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years. In like fashion, anindividual may be made less susceptible to an inflammatory conditionthat is expected to occur as a result of some medical treatment, e.g.,graft versus host disease resulting from the transplantation of cells,tissue or an organ into the individual.

[0207] The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer or metered dose inhaler; intratracheal, intranasal, epidermaland transdermal, oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration. Oligonucleotides with at least one2′-O-methoxyethyl modification are believed to be particularly usefulfor oral administration.

[0208] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

[0209] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Compositions for oraladministration also include pulsatile delivery compositions andbioadhesive composition as described in copending U.S. patentapplication Ser. No. 09/944,493, filed Aug. 22, 2001, and Ser. No.09/935,316, filed Aug. 22, 2001, the entire disclosures of which areincorporated herein by reference.

[0210] Compositions for parenteral, intrathecal or intraventricularadministration may include sterile aqueous, solutions which may alsocontain buffers, diluents and other suitable additives.

[0211] Dosing is dependent on severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual oligonucleotides, andcan generally be estimated based on EC₅₀s found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years.

[0212] The following examples illustrate the invention and are notintended to limit the same. Those skilled in the art will recognize, orbe able to ascertain through routine experimentation, numerousequivalents to the specific substances and procedures described herein.Such equivalents are considered to be within the scope of the presentinvention.

[0213] The following examples are provided for illustrative purposesonly and are not intended to limit the invention.

EXAMPLES Example 1 Synthesis of Nucleic Acids Oligonucleotides

[0214] Oligonucleotides were synthesized on an automated DNA synthesizerusing standard phosphoramidite chemistry with oxidation using iodine.β-Cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

[0215] The 2′-fluoro phosphorothioate oligonucleotides of the inventionwere synthesized using 5′-dimethoxytrityl-3′-phosphoramidites andprepared as disclosed in U.S. patent application Ser. No. 463,358, filedJan. 11, 1990, and Ser. No. 566,977, filed Aug. 13, 1990, which areassigned to the same assignee as the instant application and which areincorporated by reference herein. The 2′-fluoro oligonucleotides wereprepared using phosphoramidite chemistry and a slight modification ofthe standard DNA synthesis protocol: deprotection was effected usingmethanolic ammonia at room temperature.

[0216] The 2′-methoxy (2′-O-methyl) oligonucleotides of the inventionwere synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base is increased to 360 seconds.Other 2′-alkoxy oligonucleotides are synthesized by a modification ofthis method, using appropriate 2′-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va. The 3′-base used tostart the synthesis was a 2′-deoxyribonucleotide. The 2′-O-propyloligonucleotides of the invention are prepared by a slight modificationof this procedure.

[0217] The 2′ methoxyethoxy (2′-O—CH₂CH₂OCH₃) oligonucleotides of theinvention were synthesized according to the method of Martin, Helv.Chim. Acta 1995, 78, 486. For ease of synthesis, the last nucleotide wasa deoxynucleotide. All 2′-O—CH₂CH₂OCH₃ cytosines were 5-methylcytosines, which were synthesized according to the following procedures.

[0218] Synthesis of 5-Methyl Cytosine Monomers:2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

[0219] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid which was crushed to a light tan powder(57 g, 85% crude yield). The material was used as is for furtherreactions.

[0220] 2′-O-Methoxyethyl-5-methyluridine

[0221] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product.

[0222] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

[0223]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0224] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tic by first quenching the tic sample with the additionof MeOH. Upon completion of the reaction, as judged by tic, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1Y. Pure product fractions were evaporatedto yield 96 g (84%).

[0225] 3t-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0226] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added to the later solutiondropwise, over a 45 minute period. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

[0227] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0228] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0229]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0230] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tlc showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0231]N⁴-Benzoyl-21-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0232]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and-2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (tlc showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄; and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g.(87%) of the title compound.

[0233] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites:2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0234] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0235] 5′-O-tert-Butyldiphenylsilyl-O₂-2′-anhydro-5-methyluridine

[0236] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.669, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

[0237]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0238] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for are-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0239]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0240]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethylazodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (6.0:40), to get2′([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

[0241]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0242]2′-O-(.[2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0243] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5

[0244]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 100 C under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na2SO4 and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

[0245] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0246] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂).<Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

[0247] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0248] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg; 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0249]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0250] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P205 under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N1,N1-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO3 (40 mL). Ethyl acetate layer was dried over anhydrousNa2SO4 and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

[0251] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0252] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0253]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0254] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-α-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(PCT WO94/02501). Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,1-4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0255] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0256] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0257] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0258] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O2-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155 C for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

[0259]5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methylUridine

[0260] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO3solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et3N (20:1, v/v, with 1% triethylamine)gives the title compound.

[0261]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O— (cyanoethyl-N,N-diisopropyl)phosphoramidite

[0262] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

[0263] Purification:

[0264] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothioate analogs were judged from electrophoresis to be greaterthan 80% full length material.

[0265] B7 Antisense Oligonucleotides

[0266] A series of oligonucleotides with sequences designed to hybridizeto the published human B7-1 (hB7-1) and murine (mB7-1) mRNA sequences(Freeman et al., J. Immunol., 1989, 143, 2714, and Freeman et al., J.Exp. Med., 1991, 174, 625 respectively). The sequences of andmodifications to these oligonucleotides, and the location of each oftheir target sites on the hB7-1 mRNA, are given in Tables 1 and 2.Similarly, a series of oligonucleotides with sequences designed tohybridize to the human B7-2 (hB7-2) and murine B7-2 (mB7-2) mRNApublished sequences (respectively, Azuma et al., Nature, 1993, 366, 76;Chen et al., J. Immunol., 1994, 152, 4929 were synthesized. Thesequences of and modifications to these oligonucleotides and thelocation of each of their target sites on the hB7-2 mRNA are describedin Tables 3 and 4. Antisense oligonucleotides targeted to ICAM-1,including ISIS 2302 (SEQ ID NO: 17), have been described in U.S. Pat.No. 5,514,788, which issued May 7, 1996, hereby incorporated byreference. ISIS 1082 (SEQ ID NO: 102) and ISIS 3082 (SEQ ID NO: 101)have been previously described (Stepkowski et al., J. Immunol., 1994,153, 5336).

[0267] Subsequent to their initial cloning, alternative splicing eventsof B7 transcripts have been reported. The reported alternative splicingfor B7-1 is relatively simple, in that it results in messages extended5′ relative to the 5′ terminus of the human and murine B7-1 cDNAsequences originally reported (Borriello et al., J. Immunol., 1994, 153,5038; Inobe et al., J. Immunol., 1996, 157, 588). In order to retain thenumbering of the B7-1 sequences found in the references initiallyreporting B7-1 sequences, positions within these 5′ extensions of theinitially reported sequences have been given negative numbers (beginningwith position −1, the most 3′ base of the 5′ extension) in Tables 1 and2. The processing of murine B7-2 transcripts is considerably morecomplex than that so far reported for B7-1; for example, at least fivedistinct murine B7-2 mRNAs, and at least two distinct human B7-2 mRNAs,can be produced by alternative splicing events (Borriello et al., J.Immunol., 1995, 155, 5490; Freeman et al., WO 95/03408, published Feb.2, 1995; see also Jellis et al., Immunogenet., 1995, 42, 85). The natureof these splicing events is such that different 5′ exons are used toproduce distinct B7-2 mRNAs, each of which has a unique 5′ sequence butwhich share a 3′ portion consisting of some or all of the B7-2 sequenceinitially reported. As a result, positions within the 5′ extensions ofB7-2 messages cannot be uniquely related to a position within thesequence initially reported. Accordingly, in Table 3, a different set ofcoordinates (corresponding to those of SEQ ID NO: 1 of WO 95/03408) and,in Table 4, the exon number (as given in Borriello et al., J. Immunol.,1995, 155, 5490) is used to specify the location of targeted sequenceswhich are not included in the initially reported B7-2 sequence.Furthermore, although these 5′ extended messages contain potentialin-frame start codons upstream from the ones indicated in the initiallypublished sequences, for simplicity's sake, such additional potentialstart codons are not indicated in the description of target sites inTables 1-4.

[0268] In Tables 1-4, the following abbreviations are used: UTR,untranslated region; ORF, open reading frame; tIR, translationinitiation region; tTR, translation termination region; FITC,fluorescein isothiocyanate. Chemical modifications are indicated asfollows. Residues having 2′fluoro (2° F.), 2′-methoxy (2′MO) or2′-methoxyethoxy (2′ME) modification are emboldened, with the type ofmodification being indicated by the respective abbreviations. Unlessotherwise indicated, interresidue linkages are phosphodiester linkages;phosphorothioate linkages are indicated by an “S” in the superscriptposition (e.g., T^(S)A). Target positions are numbered according toFreeman et al., J. Immunol., 1989, 143:2714 (human B7-1 cDNA sequence;Table l), Freeman et al., J. Exp. Med., 1991, 174, 625 (murine B7-1 cDNAsequence; Table 2), Azuma et al., Nature, 1993, 366:76 (human B7-2 cDNAsequence; Table 3) and Chen et al., J. Immunol., 1994, 152:4929 (murineB7-2 cDNA sequence; Table 4). Nucleotide base codes are as given in 37C.F.R. § 1.822(b)(1). TABLE 1 Sequences of Oligonucleotides Targeted toHuman B7-1 mRNA SEQ Target Position; Site OligonucleotideSequence(5′−>3′) and ID ISIS # (and/or Description) ChemicalModifications NO: 13797 0053-0072; 5′ UTRG^(S)G^(S)G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)T^(S)C^(S)T^(S)G^(S)A22 13798 0132-0151; 5′ UTRG^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)G^(S)T^(S)T^(S)G^(S)T^(S)G^(S)G^(S)A23 13799 0138-0157; 5′ UTRG^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)G^(S)T24 13800 0158-0177; 5′ UTRA^(S)C^(S)A^(S)C^(S)A^(S)C^(S)A^(S)G^(S)A^(S)G^(S)A^(S)T^(S)T^(S)G^(S)G^(S)A^(S)G^(S)G^(S)G^(S)T25 13801 0193-0212; 5′ UTRG^(S)C^(S)T^(S)C^(S)A^(S)C^(S)G^(S)T^(S)A^(S)G^(S)A^(S)G^(S)A^(S)C^(S)C^(S)C^(S)T^(S)C^(S)C26 13802 0217-0236; 5′ UTRG^(S)G^(S)C^(S)A^(S)G^(S)G^(S)G^(S)C^(S)T^(S)G^(S)A^(S)T^(S)G^(S)A^(S)C^(S)A^(S)A^(S)T^(S)C^(S)C27 13803 0226-0245; 5′ UTRT^(S)G^(S)C^(S)A^(S)A^(S)A^(S)A^(S)C^(S)A^(S)G^(S)G^(S)C^(S)A^(S)G^(S)G^(S)G^(S)C^(S)T^(S)G^(S)A28 13804 0246-0265; 5′ UTRA^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S)G^(S)G^(S)C^(S)A^(S)C^(S)T^(S)T^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G29 13805 0320-0339;tIRC^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T^(S)C^(S)C^(S)G^(S)T^(S)G^(S)T^(S)G^(S)T^(S)G^(S)G^(S)C^(S)C^(S)C30 13806 0380-0399; 5′ ORFG^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)C^(S)C^(S)A^(S)A^(S)G^(S)A^(S)G^(S)C31 13807 0450-0469; 5′ ORFC^(S)C^(S)A^(S)C^(S)A^(S)G^(S)G^(S)A^(S)C^(S)A^(S)G^(S)C^(S)G^(S)T^(S)T^(S)G^(S)C^(S)C^(S)A^(S)C32 13808 0568-0587; 5′ ORFC^(S)C^(S)G^(S)G^(S)T^(S)T^(S)C^(S)T^(S)T^(S)G^(S)T^(S)A^(S)C^(S)T^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C33 13809 0634-0653; central ORFG^(S)C^(S)C^(S)C^(S)T^(S)C^(S)G^(S)T^(S)C^(S)A^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)C^(S)G^(S)C^(S)A51 13810 0829-0848; central ORFC^(S)C^(S)A^(S)A^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)G^(S)A^(S)G^(S)G^(S)T^(S)G^(S)A^(S)G^(S)G^(S)C34 13811 1102-1121; 3′ORFG^(S)G^(S)C^(S)A^(S)A^(S)A^(S)G^(S)C^(S)A^(S)G^(S)T^(S)A^(S)G^(S)G^(S)T^(S)C^(S)A^(S)G^(S)C35 13812 1254-1273; 3′-UTRG^(S)C^(S)C^(S)T^(S)C^(S)A^(S)T^(S)G^(S)A^(S)T^(S)C^(S)C^(S)C^(S)C^(S)A^(S)C^(S)G^(S)A^(S)T^(S)C36 13872 (scrambled#13812)A^(S)G^(S)T^(S)C^(S)C^(S)T^(S)A^(S)C^(S)T^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)C^(S)C^(S)C^(S)T52 12361 0056-0075; 5′ UTRT^(S)C^(S)A^(S)G^(S)G^(S)G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)T^(S)C38 12348 0056-0075; 5′ UTR T C A G G G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S) C ^(S) C A C T T C 38 (2′ME)12473 0056-0075; 5′ UTR T ^(S) C ^(S) A ^(S) G ^(S) G ^(S) G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)C^(S)T^(S)C^(S) C ^(S) A ^(S) C ^(S) T ^(S)T ^(S) C 38 (2′ F1) 12362 0143-0162; 5′ UTRA^(S)G^(S)G^(S)G^(S)T^(S)G^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)C^(S)A39 12349 0143-0162; 5′ UTR A G G G T G^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S) G ^(S) T C T C C A 39 (2′ ME)12474 0143-0162; 5′ UTR A ^(S) G ^(S) G ^(S) G ^(S) T ^(S) G^(S)T^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S) T ^(S) C ^(S) T ^(S) C ^(S)C ^(S)A 39 (2′ F1) 12363 0315-0334;tIRC^(S)T^(S)C^(S)C^(S)G^(S)T^(S)G^(S)T^(S)G^(S)T^(S)G^(S)G^(S)C^(S)C^(S)C^(S)A^(S)T^(S)G^(S)G^(S)C40 12350 0315-0334; tIR C T C C G T^(S)G^(S)T^(S)G^(S)T^(S)G^(S)G^(S)C^(S) C C A T G G C 40 (2′ ME) 124750315-0334; tIR C ^(S) T ^(S) C ^(S) C ^(S) G ^(S) T^(S)G^(S)T^(S)G^(S)G^(S)C^(S)C^(S) C ^(S) A ^(S) T ^(S) G ^(S) G ^(S) C40 (2′ F1) 12364 0334-0353; 5′ ORFG^(S)G^(S)A^(S)T^(S)G^(S)G^(S)T^(S)G^(S)A^(S)T^(S)G^(S)T^(S)T^(S)C^(S)C^(S)C^(S)T^(S)G^(S)C^(S)C41 12351 0334-0353; 5′ ORF G G A T G G^(S)T^(S)G^(S)A^(S)T^(S)G^(S)T^(S)T^(S) C C C T G C C 41 (2′ ME) 124760334-0353; 5′ ORF G ^(S) G ^(S) A ^(S) T ^(S) G ^(S) G^(S)T^(S)G^(S)A^(S)T^(S)G^(S)T^(S)T^(S)C^(S) C ^(S) C ^(S) T ^(S) G ^(S)C ^(S) C 41 (2′ F1) 12365 0387-0406; 5′ ORFT^(S)G^(S)A^(S)G^(S)A^(S)A^(S)A^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)C42 12352 0387-0406; 5′ ORF T G A G A A^(S)A^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S) C ^(S) C A G C A C 42 (2′ ME)12477 0387-0406; 5′ ORF T ^(S) G ^(S) A ^(S) G ^(S) A ^(S) A^(S)A^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S)C^(S) C ^(S) A ^(S) G ^(S) C ^(S)A ^(S) C 42 (2′ F1) 12366 0621-0640; central ORFG^(S)G^(S)G^(S)C^(S)G^(S)C^(S)A^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)C^(S)A^(S)C43 12353 0621-0640; central ORFG^(S)G^(S)G^(S)C^(S)G^(S)C^(S)A^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)C^(S)A^(S)C43 (2′ ME) 12478 0621-0640; central ORF G ^(S) G ^(S) G ^(S) C ^(S) G^(S) C ^(S)A^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S) G ^(S) A ^(S) T ^(S)C ^(S) A ^(S) C 43 (2′ F1) 12367 1042-1061; 3′ORFG^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)A^(S)G^(S)C^(S)A^(S)G^(S)G^(S)T44 12354 1042-1061; 3′ORF G G C C C A^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S) A G C A G G T 44 (2′ ME) 124791042-1061; 3′ORF G ^(S) G ^(S) C ^(S) C ^(S) C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)A^(S) G C A G G T 44 (2′ F1)12368 1069-1088; tTRA^(S)G^(S)G^(S)G^(S)C^(S)G^(S)T^(S)A^(S)C^(S)A^(S)C^(S)T^(S)T^(S)T^(S)C^(S)C^(S)C^(S)T^(S)T^(S)C45 12355 1069-1088; tTR A G G G C G^(S)T^(S)A^(S)C^(S)A^(S)C^(S)T^(S)T^(S) T C C C T T C 45 (2′ ME) 124801069-1088; tTR A ^(S) G ^(S) G ^(S) G ^(S) C ^(S) G^(S)T^(S)A^(S)C^(S)A^(S)C^(S)T^(S)T^(S)T^(S) C ^(S) C ^(S) C ^(S) T ^(S)T ^(S) C 45 (2′ F1) 12369 1100-1209; tTRC^(S)A^(S)G^(S)C^(S)C^(S)C^(S)C^(S)T^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S)T^(S)G^(S)C^(S)G^(S)G^(S)A46 12356 1100-1209; tTR C A G C C C^(S)C^(S)T^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S) T G C G G A 46 (2′ ME)12481 1100-1209; tTR C ^(S) A ^(S) G ^(S) C ^(S) C ^(S) C^(S)C^(S)T^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S) T ^(S) G ^(S) C ^(S) G ^(S)G ^(S) A 46 (2′ F1) 12370 1360-1380; 3′ UTRA^(S)A^(S)G^(S)G^(S)A^(S)G^(S)A^(S)G^(S)G^(S)G^(S)A^(S)T^(S)G^(S)C^(S)C^(S)A^(S)G^(S)C^(S)C^(S)A47 12357 1360-1380; 3′ UTR AAGGAGSASGSGSGSASTSOSCCAGCCA 47 (2′ ME) 124821360-1380; 3′ UTR A ^(S) A ^(S) G ^(S) G ^(S) A ^(S) G^(S)A^(S)G^(S)G^(S)G^(S)A^(S)T^(S)G^(S)C^(S) C ^(A) G ^(S) C ^(S) C ^(S)A 47 (2′ F1) 12914 (−0038 to −0059; 5′  C ^(S) T ^(S) G ^(S) T ^(S) T^(S) A ^(S) C ^(S) T ^(S) T ^(S) T ^(S) A ^(S) C ^(S) A ^(S) G ^(S) A^(S) G ^(S) G ^(S) G ^(S) T ^(S) T ^(S) T ^(S) G 48 UTR of alternative(2′ MO) mRNA) 12915 (−0035 to −0059; 5′  C ^(S) T ^(S) T ^(S) C ^(S) T^(S) G ^(S) T ^(S) T ^(S) A ^(S) C ^(S) T ^(S) T ^(S) T ^(S) A ^(S) C^(S) A ^(S) G ^(S) A ^(S) G ^(S) G ^(S) G ^(S) T ^(S) 49 UTR ofalternative T ^(S) T ^(S) G mRNA) (2′ ME) 13498 (−0038 to −0058; 5′  C^(S) T ^(S) G ^(S) T ^(S) T ^(S) A ^(S) C ^(S) T ^(S) T ^(S) T ^(S) A^(S) C ^(S) A ^(S) G ^(S) A ^(S) G ^(S) G ^(S) G ^(S) T ^(S) T ^(S) T 50UTR of alternative (2′ ME) mRNA) 13499 (−0038 to −0058; 5′  C T G T T AC T T T A C A G A G G G T T T 50 UTR of alternative (2′ ME) mRNA)

[0269] TABLE 2 Sequences of Oligonucleotides Targeted to Murine B7-1mRNA SEQ Oligonucleotide Sequence (5′−>3′) ID ISIS # Target Position;Site and Chemical Modifications NO: 14419 0009-0028; 5′ UTRA^(S)G^(S)T^(S)A^(S)A^(S)G^(S)A^(S)G^(S)T^(S)C^(S)T^(S)A^(S)T^(S)T^(S)G^(S)A^(S)G^(S)G^(S)T^(S)A53 14420 0041-0060; 5′ UTRG^(S)G^(S)T^(S)T^(S)G^(S)A^(S)G^(S)T^(S)T^(S)T^(S)C^(S)A^(S)C^(S)A^(S)A^(S)C^(S)C^(S)T^(S)G^(S)A54 14421 0071-0091; 5′ UTRG^(S)T^(S)C^(S)C^(S)A^(S)C^(S)A^(S)G^(S)A^(S)A^(S)T^(S)G^(S)G^(S)A^(S)A^(S)C^(S)A^(S)G^(S)A^(S)G55 14422 0109-0128; 5′ UTRG^(S)G^(S)C^(S)A^(S)T^(S)C^(S)C^(S)A^(S)C^(S)C^(S)C^(S)G^(S)G^(S)C^(S)A^(S)G^(S)A^(S)T^(S)G^(S)C56 14423 0114-0133; 5′ UTRT^(S)G^(S)G^(S)A^(S)T^(S)G^(S)G^(S)C^(S)A^(S)T^(S)C^(S)C^(S)A^(S)C^(S)C^(S)C^(S)G^(S)G^(S)C^(S)A57 14424 0168-0187; 5′ UTRA^(S)G^(S)G^(S)C^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C^(S)T^(S)A^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)C^(S)A58 14425 0181-0200; 5′ UTRG^(S)C^(S)C^(S)A^(S)A^(S)T^(S)G^(S)G^(S)A^(S)G^(S)C^(S)T^(S)T^(S)A^(S)G^(S)G^(S)C^(S)A^(S)C^(S)C59 14426 0208-0217; 5′ UTRC^(S)A^(S)T^(S)G^(S)A^(S)T^(S)G^(S)G^(S)G^(S)G^(S)A^(S)A^(S)A^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A60 14427 0242-0261; tIRA^(S)A^(S)T^(S)T^(S)G^(S)C^(S)A^(S)A^(S)G^(S)C^(S)C^(S)A^(S)T^(S)A^(S)G^(S)C^(S)T^(S)T^(S)C^(S)A61 14428 0393-0412; 5′ ORFC^(S)G^(S)G^(S)C^(S)A^(S)A^(S)G^(S)G^(S)C^(S)A^(S)G^(S)C^(S)A^(S)A^(S)T^(S)A^(S)C^(S)C^(S)T^(S)T62 14909 0478-0497; 5′ ORFC^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)A^(S)T^(S)G^(S)A^(S)C^(S)A^(S)G^(S)A^(S)C^(S)A^(S)G^(S)C^(S)A63 14910 0569-0588; central ORFG^(S)G^(S)T^(S)C^(S)T^(S)G^(S)A^(S)A^(S)A^(S)G^(S)G^(S)A^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C64 14911 0745-0764; central ORFT^(S)G^(S)G^(S)G^(S)A^(S)A^(S)A^(S)C^(S)C^(S)C^(S)C^(S)C^(S)G^(S)G^(S)A^(S)A^(S)C^(S)C^(S)A^(S)A65 14912 0750-0769; central ORFG^(S)G^(S)C^(S)T^(S)T^(S)T^(S)G^(S)G^(S)G^(S)A^(S)A^(S)A^(S)C^(S)C^(S)C^(S)C^(S)C^(S)G^(S)G^(S)A66 14913 0825-0844; 3′ ORFT^(S)C^(S)A^(S)G^(S)A^(S)T^(S)T^(S)C^(S)A^(S)G^(S)G^(S)A^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G^(S)A67 14914 0932-0951; 3′ ORFC^(S)C^(S)C^(S)A^(S)G^(S)G^(S)T^(S)G^(S)A^(S)A^(S)G^(S)T^(S)C^(S)C^(S)T^(S)C^(S)T^(S)G^(S)A^(S)C68 14915 1001-1020; 3′ ORFC^(S)T^(S)G^(S)C^(S)G^(S)C^(S)C^(S)G^(S)A^(S)A^(S)T^(S)C^(S)C^(S)T^(S)G^(S)C^(S)C^(S)C^(S)C^(S)A69 14916 1125-1144; tTRC^(S)A^(S)G^(S)C^(S)C^(S)C^(S)G^(S)A^(S)A^(S)G^(S)G^(S)T^(S)A^(S)A^(S)G^(S)G^(S)C^(S)T^(S)G70 14917 1229-1248; 3′ UTRT^(S)C^(S)A^(S)G^(S)C^(S)T^(S)A^(S)G^(S)C^(S)A^(S)C^(S)G^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G^(S)A^(S)A71 14918 1329-1348; 3′ UTRG^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)A^(S)A^(S)C^(S)T^(S)T^(S)G^(S)C^(S)C^(S)C^(S)G^(S)T72 14919 1377-1393; 3′ UTRC^(S)C^(S)A^(S)C^(S)C^(S)A^(S)C^(S)A^(S)G^(S)G^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)G^(S)C^(S)C73 12912 −0067 to −0049; 5′ UTR G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) T^(S) G ^(S) A ^(S) G ^(S) G ^(S) G ^(S) C ^(S) A ^(S) A ^(S) T ^(S) C^(S) T ^(S) A ^(S) A 74 (2′MO) 12913 −0067 to −0047; 5′ UTR G ^(S) T^(S) G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) T ^(S) G ^(S) A ^(S) G ^(S) G^(S) G ^(S) C ^(S) A ^(S) A ^(S) T ^(S) C ^(S) T ^(S) A ^(S) 75 A (2′MO)13496 −0067 to −0047; 5′ UTR G ^(S) T ^(S) G ^(S) G ^(S) C ^(S) C ^(S) A^(S) T ^(S) G ^(S) A ^(S) G ^(S) G ^(S) G ^(S) C ^(S) A ^(S) A ^(S) T^(S) C ^(S) T ^(S) A ^(S) 75 A (2′ME) 13497 −0067 to −0047; 5′ UTR G T GG C C A T G A G G G C A A T C T A 75 A (2′ME)

[0270] TABLE 3 Sequences of Oligonucleotides Targeted to Human B7-2 mRNASEQ ID ISIS # Target Position*; Site** Oligonucleotide Sequence (5′−>3′)NO: 9133 1367-1386; 3′-UTRT^(S)T^(S)C^(S)C^(S)A^(S)G^(S)G^(S)T^(S)C^(S)A^(S)T^(S)G^(S)A^(S)G^(S)C^(S)C^(S)A^(S)T^(S)T^(S)A3 10715 scrambled control of #9133G^(S)A^(S)T^(S)T^(S)T^(S)A^(S)A^(S)C^(S)A^(S)T^(S)T^(S)T^(S)G^(S)G^(S)C^(S)G^(S)C^(S)C^(S)C^(S)A76 9134 1333-1352; 3′-UTRC^(S)A^(S)T^(S)A^(S)A^(S)G^(S)G^(S)T^(S)G^(S)T^(S)G^(S)C^(S)T^(S)C^(S)T^(S)G^(S)A^(S)A^(S)G^(S)T^(S)G4 9135 1211-1230; 3′-UTRT^(S)T^(S)A^(S)C^(S)T^(S)C^(S)A^(S)T^(S)G^(S)G^(S)T^(S)A^(S)A^(S)T^(S)G^(S)T^(S)C^(S)T^(S)T^(S)T^(S)5 9136 1101-1120; tTRA^(S)T^(S)T^(S)A^(S)A^(S)A^(S)A^(S)A^(S)C^(S)A^(S)T^(S)G^(S)T^(S)A^(S)T^(S)C^(S)A^(S)C^(S)T^(S)T^(S)6 10716 (scrambled#9136)A^(S)A^(S)A^(S)G^(S)T^(S)T^(S)A^(S)C^(S)A^(S)A^(S)C^(S)A^(S)T^(S)T^(S)A^(S)T^(S)A^(S)T^(S)C^(S)T77 9137 0054-0074; 5′-UTRG^(S)G^(S)A^(S)A^(S)C^(S)A^(S)C^(S)A^(S)G^(S)A^(S)A^(S)G^(S)C^(S)A^(S)A^(S)G^(S)G^(S)T^(S)G^(S)C^(S)T7 9138 0001-0020; 5′-UTRC^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C^(S)T^(S)A^(S)A^(S)G^(S)G^(S)C^(S)T^(S)C^(S)C^(S)T8 9139 0133-0152; tIRC^(S)C^(S)A^(S)T^(S)A^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G^(S)T^(S)C^(S)A^(S)C^(S)A^(S)A^(S)A^(S)T9 10877 (scrambled #9139)A^(S)G^(S)T^(S)G^(S)C^(S)G^(S)A^(S)T^(S)T^(S)C^(S)T^(S)C^(S)A^(S)A^(S)A^(S)C^(S)C^(S)T^(S)A^(S)C^(S)78 10367 0073-0092; 5′-UTRG^(S)C^(S)A^(S)C^(S)A^(S)G^(S)C^(S)A^(S)G^(S)C^(S)A^(S)T^(S)T^(S)C^(S)C^(S)C^(S)A^(S)A^(S)G^(S)G^(S)G10 10368 0240-0259; 5′ORFT^(S)T^(S)G^(S)C^(S)A^(S)A^(S)A^(S)T^(S)T^(S)G^(S)G^(S)C^(S)A^(S)T^(S)G^(S)G^(S)C^(S)A^(S)G^(S)G11 10369 1122-1141; 3′-UTRT^(S)G^(S)G^(S)T^(S)A^(S)T^(S)G^(S)G^(S)G^(S)C^(S)T^(S)T^(S)T^(S)A^(S)C^(S)T^(S)C^(S)T^(S)T^(S)T12 10370 1171-1190; 3′-UTRA^(S)A^(S)A^(S)A^(S)G^(S)G^(S)T^(S)T^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G^(S)A^(S)A^(S)C^(S)G^(S)G13 10371 1233-1252; 3′-UTRG^(S)G^(S)G^(S)A^(S)G^(S)T^(S)C^(S)C^(S)T^(S)G^(S)G^(S)A^(S)G^(S)C^(S)C^(S)C^(S)C^(S)C^(S)T^(S)T14 10372 1353-1372; 3′-UTRC^(S)A^(S)T^(S)T^(S)A^(S)A^(S)G^(S)C^(S)T^(S)G^(S)G^(S)G^(S)C^(S)T^(S)T^(S)G^(S)G^(S)C^(S)C15 11149 0019-0034; 5′-UTRT^(S)A^(S)T^(S)T^(S)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C79 11151 0020-0034; 5′-UTRT^(S)A^(S)T^(S)T^(S)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C80 11150 0021-0034; 5′-UTRT^(S)A^(S)T^(S)T^(S)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S); C 8110373 0011-0030; 5′-UTRT^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C16 10721 (scrambled #10373)C^(S)G^(S)A^(S)C^(S)A^(S)G^(S)G^(S)C^(S)T^(S)C^(S)C^(S)T^(S)G^(S)C^(S)T^(S)C^(S)C^(S)T^(S)C82 10729 (5′FITC#10373)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C16 10782 (5′cholesterol#10373)T^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C16 #10373 Deletion Derivatives: 10373 0011-0030; 5′-UTRT^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C16 10888 0011-0026; 5′-UTRA^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C83 10889 0015-0030; 5′-UTRT^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C84 10991 0015-0024; 5′-UTRC^(S)T^(S)C^(S)C^(S)C^(S)G^(S)G^(S)T^(S)A^(S)C 85 10992 0015-0025;5′-UTR G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 86 109930015-0026; 5′-UTRA^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 87 109940015-0027; 5′-UTRG^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 88 109950015-0028; 5′-UTRC^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C 8910996 0015-0029; 5′-UTRG^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C90 11232 0017-0029; 5′-UTRG^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T 92 #10996Derivatives: 10996 0015-0029; 5′-UTRG^(S)C^(S)G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S)T^(S)A^(S)C90 11806 (scrambled#10996)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)A^(S)A^(S)G^(S)T^(S)C^(S)T92 11539 (fully 2′ MO #10996) G ^(S) C ^(S) G ^(S) A ^(S) G ^(S) C ^(S)T ^(S) C ^(S) C ^(S) C ^(S) C ^(S) G ^(S) T ^(S) A ^(S)C (2′MO) 90 11540(control for #11539) G ^(S) C ^(S) C ^(S) G ^(S) C ^(S) C ^(S) G ^(S) C^(S) C ^(S) A ^(S) A ^(S) G ^(S) T ^(S) C ^(S)T (2′MO) 92 11541 (#109967-base “gapmer”) G ^(S) C ^(S) G ^(S) A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S) G ^(S) T ^(S) A ^(S) C (2′MO) 9011542 (control for #11541) G ^(S) C ^(S) C ^(S) G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)A^(S)A^(S) G ^(S) T ^(S) C ^(S) T (2′MO) 9211543 (#10996 9-base “gapmer”) G ^(S) C ^(S) G^(S)A^(S)G^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)G^(S) T ^(S) A ^(S) C (2′MO)90 11544 (control for #11543) G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)A^(S)A^(S)G^(S)T^(S)C^(S)T(2′MO) 92 12892 0001-0020 C ^(S) C ^(S) G ^(S) T ^(S) A ^(S) C ^(S) C^(S) T ^(S) C ^(S) C ^(S) T ^(S) A ^(S) A ^(S) G ^(S) G ^(S) T ^(S) C^(S) C 98 (2′MO)

[0271] TABLE 4 Sequences of oligonucleotides Targeted to Murine B7-2mRNA ISIS # Target Position; Site Oligonucleotide Sequence (5′−>3′) SEQID NO: 11347 1094-1113; 3′ UTRA^(S)A^(S)A^(S)T^(S)T^(S)C^(S)C^(S)A^(S)A^(S)T^(S)C^(S)A^(S)G^(S)C^(S)T^(S)G^(S)A^(S)G^(S)A121 11348 1062-1081; 3′ UTRT^(S)C^(S)T^(S)G^(S)A^(S)G^(S)A^(S)A^(S)A^(S)C^(S)T^(S)C^(S)T^(S)G^(S)A^(S)T^(S)T^(S)C122 11349 1012-1031; 3′ UTRT^(S)C^(S)C^(S)T^(S)C^(S)A^(S)G^(S)G^(S)C^(S)T^(S)C^(S)T^(S)C^(S)A^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T123 11350 0019-1138; 5′ UTRG^(S)G^(S)T^(S)T^(S)G^(S)T^(S)T^(S)C^(S)A^(S)A^(S)G^(S)T^(S)C^(S)C^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G124 11351 0037-0056; 5′ UTRA^(S)C^(S)A^(S)C^(S)G^(S)T^(S)C^(S)A^(S)C^(S)A^(S)G^(S)G^(S)A^(S)G^(S)T^(S)C^(S)T^(S)G^(S)G103 11352 0089-0108; tIRC^(S)A^(S)A^(S)G^(S)C^(S)C^(S)C^(S)A^(S)T^(S)G^(S)G^(S)T^(S)G^(S)C^(S)A^(S)T^(S)C^(S)T^(S)G^(S)G104 11353 0073-0092; tIRC^(S)T^(S)G^(S)G^(S)G^(S)G^(S)T^(S)C^(S)C^(S)A^(S)T^(S)C^(S)G^(S)T^(S)G^(S)G^(S)G^(S)T^(S)G^(S)C105 11354 0007-0026; 5′ UTRC^(S)C^(S)G^(S)T^(S)G^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T^(S)A^(S)C^(S)A^(S)G^(S)G^(S)A^(S)G^(S)C^(S)C106 11695 0058-0077; 5′ UTRG^(S)G^(S)T^(S)G^(S)C^(S)T^(S)T^(S)C^(S)C^(S)G^(S)T^(S)A^(S)A^(S)G^(S)T^(S)T^(S)C^(S)T^(S)G^(S)G107 11696 0096-0117; tTRG^(S)G^(S)A^(S)T^(S)T^(S)G^(S)C^(S)C^(S)A^(S)A^(S)G^(S)C^(S)C^(S)C^(S)A^(S)T^(S)G^(S)G^(S)T^(S)G108 11866 (scrambled #11696)C^(S)T^(S)A^(S)A^(S)G^(S)T^(S)A^(S)G^(S)T^(S)G^(S)C^(S)T^(S)A^(S)G^(S)C^(S)C^(S)G^(S)G^(S)G^(S)A109 11697 0148-0167; 5′ ORFT^(S)G^(S)C^(S)G^(S)T^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)G^(S)G^(S)A^(S)A^(S)A^(S)C^(S)A^(S)G^(S)C110 11698 0319-0338; 5′ ORFG^(S)T^(S)G^(S)C^(S)G^(S)G^(S)C^(S)C^(S)C^(S)A^(S)G^(S)G^(S)T^(S)A^(S)C^(S)T^(S)T^(S)G^(S)G^(S)C111 11699 0832-0851; 3′ ORFA^(S)C^(S)A^(S)A^(S)G^(S)G^(S)A^(S)G^(S)G^(S)A^(S)G^(S)G^(S)G^(S)C^(S)C^(S)A^(S)C^(S)A^(S)G^(S)T112 11700 0753-0772; 3′ ORFT^(S)G^(S)A^(S)G^(S)A^(S)G^(S)G^(S)T^(S)T^(S)T^(S)G^(S)G^(S)A^(S)G^(S)G^(S)A^(S)A^(S)A^(S)T^(S)C113 11701 0938-0957; 3′ ORFG^(S)A^(S)T^(S)A^(S)G^(S)T^(S)C^(S)T^(S)C^(S)T^(S)C^(S)T^(S)G^(S)T^(S)C^(S)A^(S)G^(S)C^(S)G^(S)T114 11702 0890-0909; 3′ ORFG^(S)T^(S)T^(S)G^(S)C^(S)T^(S)G^(S)G^(S)G^(S)C^(S)C^(S)T^(S)G^(S)C^(S)T^(S)A^(S)G^(S)G^(S)C^(S)T115 11865 (scrambled #11702)C^(S)T^(S)A^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)G^(S)T^(S)C^(S)G^(S)T^(S)C^(S)G^(S)G^(S)T^(S)G^(S)G116 11703 1003-1022; tIRT^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)G^(S)C^(S)C^(S)T^(S)T^(S)C^(S)A^(S)C^(S)C^(S)T^(S)C^(S)T^(S)G^(S)C117 13100 Exon 1 (Borriello et al., J. G ^(S) T ^(S) A ^(S) C ^(S) C^(S) A ^(S) G ^(S) A ^(S) T ^(S) G ^(S) A ^(S) A ^(S) G ^(S) G ^(S) T^(S) T ^(S) A ^(S) T ^(S) C ^(S) A ^(S) A 118 Immun., 1995, 155, 5490;(2′MO) 5′ UTR of alternative mRNA) 13101 Exon 4 (Borriello et al.; C^(S) T ^(S) T ^(S) T ^(S) G ^(S) G ^(S) A ^(S) G ^(S) A ^(S) T ^(S) T^(S) A ^(S) T ^(S) T ^(S) C ^(S) G ^(S) A ^(S) G ^(S) T ^(S) T 1195′ UTR of alternative mRNA) (2′MO) 13102 Exon 5 (Borriello et al.; G^(S) C ^(S) A ^(S) A ^(S) G ^(S) T ^(S) G ^(S) T ^(S) A ^(S) A ^(S) A^(S) G ^(S) C ^(S) C ^(S) C ^(S) T ^(S) G ^(S) A ^(S) G ^(S) T 1205′ UTR of alternative mRNA) (2′MO)

[0272] cDNA Clones:

[0273] A cDNA encoding the sequence for human B7-1 was isolated by usingthe reverse transcription/polymerase chain reaction (RT-PCR). Poly A+RNA from Daudi cells (ATCC accession No. CCL 213) was reversetranscribed using oligo-dT primer under standard conditions. Following a30 minute reaction at 42° C. and heat inactivation, the reaction mixture(20 μL) was brought to 100 μL with water. A 10 μL aliquot from the RTreaction was then amplified in a 50 μL PCR reaction using the 5′ primer,5′-GAT-CAG-GGT-ACC-CCA-AAG-AAA-AAG-TGA-TTT-GTC- (sense, SEQ ID NO: 20)ATT-GC-3′, and the 3′ primer,5′-GAT-AGC-CTC-GAG-OAT--AAT-GAA-TTG-GCT-GAC-AAG- (antisense, SEQ ID NO:21) AC-3′

[0274] The primers included unique restriction sites for subcloning ofthe PCR product into the vector pcDNA-3 (Invitrogen, San Diego, Calif.).The 5′ primer was designed to have identity with bases 1 to 26 of thepublished human B7-1 sequence (Freeman et al., J. Immunol., 1989, 143,2714; positions 13-38 of the primer) and includes a Kpn I restrictionsite (positions 7-12 of the primer) for use in cloning. The 3′ primerwas designed to be complementary to bases 1450 to 1471 of the publishedsequence for B7-1 (positions 14-35 of the primer) and includes a Xho Irestriction site (positions 7-12 of the primer). Following PCR, thereaction was extracted with phenol and precipitated using ethanol. Theproduct was digested with the appropriate restriction enzymes and thefull-length fragment purified by agarose gel and ligated into the vectorpcDNA-3 (Invitrogen, San Diego, Calif.) prepared by digesting with thesame enzymes. The resultant construct, pcB7-1, was confirmed byrestriction mapping and DNA sequence analysis using standard procedures.A mouse B7-1 clone, pcmB7-1, was isolated in a similar manner by RT-PCRof RNA isolated from a murine B-lymphocyte cell line, 70Z3.

[0275] A cDNA encoding the sequence for human B7-2, position 1 to 1391,was also isolated by RT-PCR. Poly A+ RNA from Daudi cells (ATCCaccession No. CCL 213) was reverse transcribed using oligo-dT primerunder standard conditions. Following a 30 minute reaction at 42° C. andheat inactivation, the reaction mixture (20 μL) was brought to 100 μLwith water. A 10 μL aliquot from the RT reaction was then amplified in a50 μL PCR reaction using the 5′ primer,5′-GAT-CAG-GGT-ACC-AGG-AGC-CTT-AGG-AGG-TAC-GG-3′, (sense, ^(S)EQ IDNO: 1) and the 3′ primer,5′-GAT-AGC-CTC-GAG-TTA-TTT-CCA-GGT-CAT-GAG-CCA-3′. (antisense, ^(S)EQ IDNO: 2)

[0276] The 5′ primer was designed to have identity with bases 1-20 ofthe published B7-2 sequence (Azuma et al., Nature, 1993, 366, 76 andGenbank Accession No. L25259; positions 13-32 of the primer) andincludes a Kpn I site (positions 7-12 of the primer) for use in cloning.The 3′ primer was designed to have complementarity to bases 1370-1391 ofthe published sequence for B7-2 (positions 13-33 of the primer) andincludes an Xho I restriction site (positions 7-12 of the primer).Following PCR, the reaction was extracted with phenol and precipitatedusing ethanol. The product was digested with Xho I and Kpn I, and thefull-length: fragment purified by agarose gel and ligated into thevector pcDNA-3 (Invitrogen, San Diego, Calif.) prepared by digestingwith the same enzymes. The resultant construct, pcB7-2, was confirmed byrestriction mapping and DNA sequence analysis using standard procedures.

[0277] A mouse B7-2 clone, pcmB7-2, was isolated in a similar manner byRT-PCR of RNA isolated from P388D1 cells using the 5′ primer,5′-GAT-CAG-GGT-ACC-AAG-AGT-GGC-TCC-TGT-AGG-CA, (sense, ^(S)EQ ID NO: 99)and the 3′ primer, 5′-GAT-AGC-CTC-GAG-GTA-GAA-TTC-CAA-TCA-GCT-GA.(antisense, ^(S)EQ ID NO: 100)

[0278] The 5′ primer has identity with bases 1-20, whereas the 3′ primeris complementary to bases 1096-1115, of the published murine B7-2sequence (Chen et al., J. Immun., 1994, 152, 4929). Both primersincorporate the respective restriction enzyme sites found in the other5′ and 3′ primers used to prepare cDNA clones. The RT-PCR product wasrestricted with Xho I and Kpn I and ligated into pcDNA-3 (Invitrogen,Carlsbad, Calif.).

[0279] Other cDNA clones, corresponding to mRNAs resulting fromalternative splicing events, are cloned in like fashion, using primerscontaining the appropriate restriction sites and having identity with(5′ primers), or complementarity to (3′ primers), the selected B7 mRNA.

Example 2 Modulation of hB7-1 Expression by Oligonucleotides

[0280] The ability of oligonucleotides to inhibit B7-1 expression wasevaluated by measuring the cell surface expression of B7-1 intransfected COS-7 cells by flow cytometry.

[0281] Methods:

[0282] A T-175 flask was seeded at 75% confluency with COS-7 cells (ATCCaccession No. CRL 1651). The plasmid pcB7-1 was introduced into cells bystandard calcium phosphate transfection. Following a 4 hourtransfection, the cells were trypsinized and seeded in 12-well dishes at80% confluency. The cells were allowed to adhere to the plastic for 1hour and were then washed with phosphate-buffered saline (PBS). OptiMEM™(GIBCO-BRL, Gaithersburg, Md.) medium was added along with 15 μg/mL ofLipofectin™ (GIBCO-BRL, Gaithersburg, Md.) and oligonucleotide at theindicated concentrations. After four additional hours, the cells werewashed with phosphate buffered saline (PBS) and incubated with fresholigonucleotide at the same concentration in DMEM (Dulbecco et al.,Virol., 1959, 8, 396; Smith et al., Virol., 1960, 12, 185) with 10%fetal calf sera (FCS).

[0283] In order to monitor the effects of oligonucleotides on cellsurface expression of B7-1, treated COS-7 cells were harvested by brieftrypsinization 24-48 hours after oligonucleotide treatment. The cellswere washed with PBS, then resuspended in 100 μL of staining buffer(PBS, 0.2% BSA, 0.1% azide) with 5 μL conjugated anti-B7-1-antibody(i.e., anti-hCD80-FITC, Ancell, Bayport, Minn., FITC: fluoresceinisothiocyanate). The cells were stained for 30 minutes at 4° C., washedwith PBS, resuspended in 300 μL containing 0.5% paraformaldehyde. Cellswere harvested and the fluorescence profiles were determined using aflow cytometer.

[0284] Results:

[0285] The oligonucleotides shown in Table 1 were evaluated, in COS-7cells transiently expressing B7-1 cDNA, for their ability to inhibitB7-1 expression. The results (FIG. 1) identified ISIS 13805, targeted tothe translation initiation codon region, and ISIS 13812, targeted to the3′ untranslated region (UTR), as the most active oligonucleotides withgreater than 50% inhibition of B7-1 expression. These oligonucleotidesare thus highly preferred. ISIS 13799.(targeted to the 5′ untranslatedregion), ISIS 13802 (targeted to the 5′ untranslated region), ISIS 13806and 13807 (both targeted to the 5′ region of the ORF), and ISIS 13810(targeted to the central portion of the ORF) demonstrated 35% to 50%inhibition of B7-1 expression. These sequences are therefore alsopreferred. Oligonucleotide ISIS 13800, which showed essentially noinhibition of B7-1 expression in the flow cytometry assay, and ISIS Nos.13805 and 13812 were then evaluated for their ability to inhibit cellsurface expression of B7-1 at various concentrations of oligonucleotide.The results of these assays are shown in FIG. 2. ISIS 13812 was asuperior inhibitor of B7-1 expression with an IC₅₀ of approximately 150nM. ISIS 13800, targeted to the 5′ UTR, was essentially inactive.

Example 3 Modulation of hB7-2 Protein by Oligonucleotides

[0286] In an initial screen, the ability of hB7-2 oligonucleotides toinhibit B7-2 expression was evaluated by measuring the cell surfaceexpression of B7-2 in transfected COS-7 cells by flow cytometry. Themethods used were similar to those given in Example 2, with theexceptions that (1) COS-7 cells were transfected with the plasmidspbcB7-2 or BBG-58, a human ICAM-1 (CD54) expression vector (R&D Systems,Minneapolis, Minn.) introduced into cells by standard calcium phosphatetransfection, (2) the oligonucleotides used were those described inTable 2, and (3) a conjugated anti-B7-2 antibody (i.e., anti-hCD86-FITCor anti-CD86-PE, PharMingen, San Diego, Calif.; PE: phycoerythrin) wasused during flow cytometry.

[0287] Results:

[0288] The results are shown in FIG. 3. At a concentration of 200 nM,ISIS 9133, ISIS 9139 and ISIS 10373 exhibited inhibitory activity of 50%or better and are therefore highly preferred. These oligonucleotides aretargeted to the 3′ untranslated region (ISIS 9133), the translationinitiation codon region (ISIS 9139) and the 5′ untranslated region (ISIS10373). At the same concentration, ISIS 10715, ISIS 10716 and ISIS10721, which are scrambled controls for ISIS 9133, ISIS 9139 and ISIS10373, respectively, showed no inhibitory activity. Treatment with ISIS10367 and ISIS 10369 resulted in greater than 25% inhibition, and theseoligonucleotides are thus also preferred. These oligonucleotides aretargeted to the 5′ (ISIS 10367) and 3′ (ISIS 10369) untranslatedregions.

Example 4 Modulation of hB7-2 mRNA by Oligonucleotides

[0289] Methods:

[0290] For ribonuclease protection assays, cells were harvested 18 hoursafter completion of oligonucleotide treatment using a Totally RNA™ kit(Ambion, Austin, Tex.). The probes for the assay were generated fromplasmids pcB7-2 (linearized by digestion with Bgl II) and pTR1-b-actin(Ambion Inc., Austin, Tex.). In vitro transcription of the linearizedplasmid from the SP6 promoter was performed in the presence of a-32P-UTP(800 Ci/mmole) yielding an antisense RNA complementary to the 3′ end ofB7-2 (position 1044-1391). The probe was gel-purified after treatmentwith DNase I to remove DNA template. Ribonuclease protection assays werecarried out using an RPA II™ kit (Ambion) according to themanufacturer's directions. Total RNA (5 μg) was hybridized overnight, at42° C., with 105 cpm of the B7-2 probe or a control beta-actin probe.The hybridization reaction was then treated, at 37⁹C for 30 minutes,with 0.4 units of RNase A and 2 units of RNase T1. Protected RNA wasprecipitated, resuspended in 10 μL of gel loading buffer andelectrophoresed on a 6% acrylamide gel with 50% w/v urea at 20 W. Thegel was then exposed and the lanes quantitated using a PhosphorImager(Molecular Dynamics, Sunnyvale, Calif.) essentially according to themanufacturer's instructions.

[0291] Results:

[0292] The extent of oligonucleotide-mediated hB7-2 mRNA modulationgenerally paralleled the effects seen for hB7-2 protein (Table 5). Aswith the protein expression (flow cytometry assays, the most activeoligonucleotides were ISIS 9133, ISIS 9139 and 10373. None of theoligonucleotides tested had an inhibitory effect on the expression of ofb-actin mRNA in the same cells. TABLE 5 Activities of OligonucleotidesTargeted to hB7-2 mRNA % Control % Control RNA ISIS NO. SEQ ID NO.Protein Expression 9133 3 70.2 46.0 9134 4 88.8 94.5 9135 5 98.2 83.49136 6 97.1 103.1 9137 7 80.5 78.1 9138 8 86.4 65.9 9139 9 47.9 32.610367 10 71.3 52.5 10368 11 81.0 84.5 10369 12 71.3 81.5 10370 13 84.383.2 10371 14 97.3 92.9 10372 15 101.7 82.5 10373 16 43.5 32.7

Example 5 Additional hB7-1 and hB7-2 Oligonucleotides

[0293] Oligonucleotides having structures and/or sequences that weremodified relative to the oligonucleotides identified during the initialscreening were prepared. These oligonucleotides were evaluated for theirability to modulate human B7-2 expression using the methods described inthe previous examples. ISIS 10996, an oligonucleotide having a 15nucleotide sequence derived from the 20 nucleotide sequence of ISIS10373, was also prepared and evaluated. ISIS 10996 comprises 15nucleotides, 51-GCG-AGC-TCC-CCG-TAC (SEQ ID NO: 90) contained within thesequence of ISIS 10373. Both ISIS 10373 and 10996 overlap a potentialstem-loop structure located within the B7-2 message comprising bases1-67 of the sequence of hB7-2 presented by Azuma et al. (Nature, 1993,366, 76). While not intending to be bound by any particular theoryregarding their mode(s) of action, ISIS 10373 and ISIS 10996 have thepotential to bind as loop 1 pseudo-half-knots at a secondary structurewithin the target RNA. U.S. Pat. No. 5,5152,438, the contents of whichare hereby incorporated by reference, describes methods for modulatinggene expression by the formation of pseudo-half-knots. Regardless oftheir mode(s) of action, despite having a shorter length than ISIS10373, the 15-mer ISIS 10996 is as (or more) active in the B7-2 proteinexpression assay than the 20-mer from which it is derived (FIG. 4; ISIS10721 is a scrambled control for ISIS 10373). A related 16-mer, ISIS10889, was also active in the B7-2 protein expression assay. However, astructurally related 14-mer (ISIS 10995), 13-mer (ISIS 10994), 12-mer(ISIS 10993), 11-mer (ISIS 10992) and 10-mer (ISIS 10991) exhibitedlittle or no activity in this assay. ISIS 10996was further derivatizedin the following ways.

[0294] ISIS 10996 derivatives having 2′ methoxethoxy substitutions wereprepared, including a fully substituted derivative (ISIS 11539),“gapmers” (ISIS 11541 and 11543) and “wingmers” (ISIS 11545 and 11547).As explained in Example 5, the 2′ methoxyethoxy substitution preventsthe action of some nucleases (e.g., RNase H) but enhances the affinityof the modified oligonucleotide for its target RNA molecule. Theseoligonucleotides are tested for their ability to modulate hB7-2 messageor function according to the methods of Examples 3, 4, 7 and 8.

[0295] ISIS 10996 derivatives were prepared in order to be evaluated fortheir ability to recruit RNase L to a target RNA molecule, e.g., hB7-2message. RNase L binds to, and is activated by, (2′-5′)(A)_(n), which isin turn produced from ATP by (2′-5′)(A)_(n) synthetase upon activationby, e.g., interferon. RNase L has been implicated in antiviralmechanisms and in the regulation of cell growth as well (Sawai, ChemicaScripta, 1986, 21, 169; Charachon et al., Biochemistry 1990, 29, 2550).The combination of anti-B7 oligonucleotides conjugated to (2′-5′)(A)_(n) is expected to result in the activation of RNase L and itstargeting to the B7 message complementary to the oligonucleotidesequence. The following oligonucleotides have identical sequences (i.e.,that of ISIS 10996) and identical (2′-5′)(A)₄ “caps” on their 5′termini: ISIS 12492, 12495, 12496 and 13107. The adenosyl residues have3′ hydroxyl groups and are linked to each other by phosphorothioatelinkages. The (3′-5′) portion of the oligonucleotide, which has asequence complementary to a portion of the human B7-2 RNA, is conjugatedto the (2′-5′)(A)₄ “cap” via a phosphorothioate linkage from the 5′residue of the (3′-5′) portion of the oligonucleotide to an n-aminohexyllinker which is bonded to the cap” via another phosphorothioate linkage.In order to test a variety of chemically diverse oligonucleotides ofthis type for their ability to recruit RNase L to a specific message,different chemical modifications were made to this set of fouroligonucleotides as follows. ISIS 12496 consists of unmodifiedoligonucleotides in the (3′-5′) portion of the oligonucleotide. In ISIS13107, phosphorothioate linkages replace the phosphate linkages found innaturally occurring nucleic acids. Phosphorothioate linkages are alsoemployed in ISIS 12492 and 12495, which additionally have2′-methoxyethoxy substitutions. These oligonucleotides are tested fortheir ability to modulate hB7-2 message or function according to themethods of Examples 3, 4, 7 and 8.

[0296] Derivatives of ISIS 109:96 having modifications at the 2′position were prepared and evaluated. The modified oligonucleotidesincluded ISIS 11539 (fully 2′-O-methyl), ISIS 11541 (having 2′-o-methyl“wings” and a central 7-base “gap”), ISIS 11543 (2′-O-methyl wings witha 9-base gap), ISIS 11545 (having a 5′ 2′-O-methyl wing) and ISIS 11547(having a 3′ 2′-O-methyl wing). The results of assays of 2′-O-methyloligonucleotides were as follows. ISIS 11539, the fully 2-O-methylversion of ISIS 10996, was not active at all in the protein expressionassay. The gapped and winged oligonucleotides (ISIS 11541, 11543, 11545and 11547) each showed some activity at 200 nM (i.e., from 0.60 to 70%expression relative to untreated cells), but less than that demonstratedby the parent compound, ISIS 10996 (i.e., about 50% expression). Similarresults were seen in RNA expression assays.

[0297] ISIS 10782, a derivative of ISIS 10373 to which cholesterol hasbeen conjugated via a 51 n-aminohexyl linker, was prepared. Lipophilicmoieties such as cholesterol have been reported to enhance the uptake bycells of oligonucleotides in some instances, although the extent towhich uptake is enhanced, if any, remains unpredictable. ISIS 10782, andother oligonucleotides comprising lipophilic moieties, are tested fortheir ability to modulate B7-2 message or function according to themethods of Examples 3, 4, 7 and 8.

[0298] A series of 2′-methoxyethoxy (herein, 2“ME”) and 2′-fluoride(herein, “2′F”) “gapmer” derivatives of the hB7-1 oligonucleotides ISIS12361 (ISIS Nos. 12348 and 12473, respectively), ISIS 12362 (ISIS Nos.12349 and 12474), ISIS 12363 (ISIS Nos. 12350 and 12475), ISIS 12364(ISIS Nos. 12351 and 12476), ISIS 12365 (ISIS Nos. 12352 and 12477),ISIS 12366 (ISIS Nos. 12353 and 12478), ISIS 12367 (ISIS Nos. 12354 and12479), ISIS 12368. (ISIS Nos. 12355 and 12480), ISIS 12369 (ISIS Nos.12356 and 12481) and ISIS 12370 (ISIS Nos. 12357 and, 12482)-wereprepared. The central, non-2′-modified portions (Agaps@) of thesederivatives support RNase H activity when the oligonucleotide is boundto its target RNA, even though the 2′-modified portions do not. However,the 2′-modified “wings” of these oligonucleotides enhance their affinityto their target RNA molecules (Cook, Chapter 9 In: Antisense Researchand Applications, Crooke et al., eds., CRC Press, Boca Raton, 1993, pp.171-172).

[0299] Another 2′ modification is the introduction of a methoxy (MO)group at this position. Like 2′ME- and 2′F-modified oligonucleotides,this modification prevents the action of RNase H on duplexes formed fromsuch oligonucleotides and their target RNA molecules, but enhances theaffinity of an oligonucleotide for its target RNA molecule. ISIS 12914and 12915 comprise sequences complementary to the 5′ untranslated regionof alternative hB7-1 mRNA molecules, which arise from alternativesplicing events of the primary hB7-1 transcript. These oligonucleotidesinclude 2′ methoxy modifications, and the enhanced target affinityresulting therefrom may allow for greater activity against alternativelyspliced B7-1 mRNA molecules which may be present in low abundance insome tissues (Inobe et al., J. Immun., 1996, 157, 582). Similarly, ISIS13498 and 13499, which comprise antisense sequences to other alternativehB7-1 mRNAs, include 2′ methoxyethoxy modifications in order to enhancetheir affinity for their target molecules, and 2′ methoxyethoxy or2′methoxy substitutions are incorporated into the hB7-2 oligonucleotidesISIS 12912, 12913, 13496 and 13497. These oligonucleotides are testedfor their ability to modulate hB7-1 essentially according to the methodsof Example 2 or hB7-2 according to the methods of Examples 3, 4, 7 and8, with the exception that, when necessary, the target cells aretransfected with a cDNA clone corresponding to the appropriatealternatively spliced B7 transcript.

Example 6 Specificity of Antisense Modulation

[0300] Several oligonucleotides of the invention were evaluated in acell surface expression flow cytometry assay to determine thespecificity of the oligonucleotides for B7-1 as contrasted with activityagainst B7-2. The oligonucleotides tested in this assay included ISIS13812, an inhibitor of B7-1 expression (FIG. 1; Example 2) and ISIS10373, an inhibitor of B7-2 expression (FIG. 3; Example 3). The resultsof this assay are shown in FIG. 5. ISIS 13812 inhibits B7-1 expressionwith little or no effect on B7-2 expression. As is also seen in FIG. 5,ISIS 10373 inhibits B7-2 expression with little or no effect on B7-1expression. ISIS 13872 (SEQ ID NO: 37, AGT-CCT-ACT-ACC-AGC-CGC-CT), ascrambled control of ISIS 13812, and ISIS 13809 (SEQ ID NO: 51) wereincluded in these assays and demonstrated essentially no activityagainst either B7-1 or B7-2.

Example 7 Modulation of hB7-2 Expression by Oligonucleotides in AntigenPresenting Cells

[0301] The ability of ISIS 10373 to inhibit expression from the nativeB7-2 gene in antigen presenting cells (APCs) was evaluated as follows.

[0302] Methods:

[0303] Monocytes were cultured and treated with oligonucleotides asfollows. For dendritic cells, EDTA-treated blood was layered ontoPolymorphprep™ (1.113 g/mL; Nycomed, Oslo, Norway) and sedimented at500×g for 30 minutes at 20° C. Mononuclear cells were harvested from theinterface. Cells were washed with PBS, with serum-free RPMI media (Mooreet al., N.Y. J. Med., 1968, 68, 2054) and then with RPMI containing 5%fetal bovine serum (FBS). Monocytes were selected by adherence toplastic cell culture cell culture dishes for 1 h at 37° C. Afteradherence, cells were treated with oligonucleotides in serum-free RPMIcontaining Lipofectin™ (8 μg/mL). After 4 hours, the cells were washed.Then RPMI containing 5% FBS and oligonucleotide was added to cells alongwith interleukin-4 (IL-4; R&D Systems, Minneapolis, Minn.) (66 ng/mL)and granulocyte-macrophage colony-stimulating factor (GM-CSF; R&DSystems, Minneapolis, Minn.) (66 ng/mL) to stimulate differentiation(Romani et al., J. Exp. Med., 1994, 180, 83, 1994). Cells were incubatedfor 48 hours, after which cell surface expression of various moleculeswas measured by flow cytometry.

[0304] Mononuclear cells isolated from fresh blood were treated witholigonucleotide in the presence of cationic lipid to promote cellularuptake. As a control oligonucleotide, ISIS 2302.(an inhibitor of ICAM-1expression; SEQ ID NO: 17) was also administered to the cells.Expression of B7-2 protein was measured by flow cytometry according tothe methods of Example 2. Monoclonal antibodies not described in theprevious Examples included anti-hCD3 (Ancell, Bayport, MN) andanti-HLA-DR (Becton Dickinson, San Jose, Calif.).

[0305] Results:

[0306] As shown in FIG. 6, ISIS 10373 has a significant inhibitoryeffect on B7-2 expression with an IC₅₀ of approximately 250 nM. ISIS10373 had only a slight effect on ICAM-1 expression even at a dose of 1μM. ISIS 2302 (SEQ ID NO: 17), a control oligonucleotide which has beenshown to inhibit ICAM-1 expression, had no effect on B7-2 expression,but significantly decreased ICAM-1 levels with an IC₅₀ of approximately250 nM. Under similar conditions, ISIS 10373 did not affect the cellsurface expression of B7-1, HLA-DR or CD3 as measured by flow cytometry.

Example 8 Modulation of T Cell Proliferation by Oligonucleotides

[0307] The ability of ISIS 2302 and ISIS 10373 to inhibit T cellproliferation was evaluated as follows. Monocytes treated witholigonucleotide and cytokines (as in Example 6) were used as antigenpresenting cells in a T cell proliferation assay. The differentiatedmonocytes were combined with CD4+ T cells from a separate donor. After48 hours, proliferation was measured by [³H] thymidine incorporation.

[0308] Methods:

[0309] For T cell proliferation assays, cells were isolated fromEDTA-treated whole blood as described above, except that a fastermigrating band containing the lymphocytes was harvested from just belowthe interface. Cells were washed as described in-Example 6 after whicherythrocytes were removed by NH₄Cl lysis. T cells were purified using aT cell enrichment column (R&D Systems, Minneapolis, Minn.) essentiallyaccording to the manufacturer's directions. CD4+ T cells were furtherenriched from the entire T cell population by depletion of CD8+ cellswith anti-CD8-conjugated magnetic beads (AMAC, Inc., Westbrook, Me.)according to the manufacturer's directions. T cells were determined tobe>80% CD4+by flow cytometry using Cy-chrome-conjugated anti-CD4 mAb(PharMingen, San Diego, Calif.).

[0310] Antigen presenting cells (APCs) were isolated as described inExample 6 and treated with mitomycin C (25 μg/mL) for 1 hour then washed3 times with PBS. APCs (10⁵ cells) were then combined with 4×10⁴ CD4+ Tcells in 350 μL of culture media. Where indicated, purified CD3 mAb wasalso added at a concentration of 1 μg/mL. During the last 6 hours of the48 hour incubation period, proliferation was measured by determininguptake of 1.5 uCi of [³H]-thymidine per well. The cells were harvestedonto filters and the radioactivity measured by scintillation counting.

[0311] Results:

[0312] As shown in FIG. 7, mononuclear cells which were notcytokine-treated slightly induced T cell proliferation, presumably dueto low levels of costimulatory molecules expressed on the cells.However, when the cells were treated with cytokines and induced todifferentiate to dendritic-like cells, expression of both ICAM-1 andB7-2 was strongly upregulated. This resulted in a strong T cellproliferative response which could be blocked with either anti-ICAM-1(ISIS 2302) or anti-B7-2 (ISIS 10373) oligonucleotides prior toinduction of the mononuclear cells. The control oligonucleotide (ISIS10721) had an insignificant effect on T cell proliferation. Acombination treatment with both the anti-ICAM-1 (ISIS 2302) andanti-B7-2 (ISIS 10373) oligonucleotides resulted in a further decreasein T cell response.

Example 9 Modulation of Murine B7 Genes by Oligonucleotides

[0313] Oligonucleotides (see Table 4) capable of inhibiting expressionof murine B7-2 transiently expressed in COS-7 cells were identified inthe following manner. A series of phosphorothioate oligonucleotidescomplementary to murine B7-2 (mB7-2) cDNA were screened for theirability to reduce mB7-2 levels (measured by flow cytometry as in Example2, except that a conjugated anti-mB7-2 antibody (i.e., anti-mCD86-PE,PharMingen, San Diego, Calif.) in COS-7 cells transfected with an mB7-2cDNA clone. Anti-mB7-2 antibody may also be obtained from the hybridomadeposited at the ATCC under accession No. HB-253. Oligonucleotides (seeTable 2) capable of modulating murine B7-1 expression are isolated inlike fashion, except that a conjugated anti-mB7-1 antibody is used inconjunction with COS-7 cells transfected with an mB7-1 cDNA clone.

[0314] For murine B7-2, the most active oligonucleotide identified wasISIS 11696 (GGA-TTG-CCA-AGC-CCA-TGG-TG, SEQ ID NO: 18), which iscomplementary to position 96-115 of the cDNA, a site which includes thetranslation initiation (AUG) codon. FIG. 8 shows a dose-response curvefor ISIS 11696 and a scrambled control, ISIS 11866(CTA-AGT-AGT-GCT-AGC-CGG-GA, SEQ ID NO: 19). ISIS 11696 inhibited cellsurface expression of B7-2 in COS-7 cells with an IC₅₀ in the range of200-300 nM, while ISIS 11866 exhibited less than 20% inhibition at thehighest concentration tested (1000 nM).

[0315] In order to further evaluate the murine B7-2 antisenseoligonucleotides, the IC-21 cell line was used. IC-21monocyte/macrophage cell line expresses both B7-1 and murine B7-2(mB7-2) constitutively. A 2-fold induction of expression can be achievedby incubating the cells in the presence of lipopolysaccharide (LPS;GIBCO-BRL, Gaithersburg, Md.) (Hathcock et al., Science, 1993, 262,905).

[0316] IC-21 cells (ATCC; accession No. TIB 186) were seeded at 80%confluency in 12-well plates in DMEM media with 10% FCS. The cells wereallowed to adhere to the plate overnight. The following day, the mediumwas removed and the cells were washed with PBS. Then 500 μL ofOPtiMEM™.(GIBCO-BRL, Gaithersburg, Md.) supplemented with 15 μg/mL ofLipofectin™ (GIBCO-BRL, Gaithersburg, Md.) was added to each well.Oligonucleotides were then added directly to the medium at the indicatedconcentrations. After incubation for 4 hours, the cells were washed withPBS and incubated overnight in culture medium supplemented with 15 μg/mLof LPS. The following day, cells were harvested by scraping, thenanalyzed for cell surface expression by flow cytometry.

[0317] ISIS 11696 and ISIS 11866 were administered to IC-21 cells in thepresence of Lipofectin™ (GIBCO-BRL, Gaithersburg, Md.). The results areshown in FIG. 9. At a concentration of 10 uM, ISIS 11696 inhibited mB7-2expression completely (and decreased mB7-2 levels below the constitutivelevel of expression), while the scrambled control oligonucleotide, ISIS11866, produced only a 40% reduction in the level of induced expression.At a concentration of 3 uM, levels of induced expression were greatlyreduced by ISIS 11696, while ISIS 11866 had little effect.

[0318] Modified oligonucleotides, comprising 2′ substitutions (e.g., 2′methoxy, 2′ methoxyethoxy) and targeted to alternative transcripts ofmurine B7-1 (ISIS 12914, 12915, 13498, 13499) or murine B7-2 (ISIS13100, 13100 and 13102) were prepared. These oligonucleotides are testedfor their ability to modulate murine B7 essentially according to theabove methods using IC-21 cells or COS-7 transfected with a cDNA clonecorresponding to the appropriate alternatively spliced B7 transcript.

Example 10 Modulation of Allograft Rejection by Oligonucleotides

[0319] A murine model for evaluating compounds for their ability toinhibit heart allograft rejection has been previouslydescribed:(Stepkowski et al., J. Immunol., 1994, 153, 5336). This modelwas used to evaluate the immunosuppressive capacity of antisenseoligonucleotides to B7 proteins alone or in combination with antisenseoligonucleotides to intercellular adhesion molecule-1 (ICAM-1).

[0320] Methods:

[0321] Heart allograft rejection studies and oligonucleotide treatmentsof BALB/c mice were performed essentially as previously described(Stepkowski et al., J. Immunol., 1994, 153, 5336). Antisenseoligonucleotides used included ISIS 11696, ISIS 3082 (targeted toICAM-1) and ISIS 1082 (a control oligonucleotide targeted to the herpesvirus UL-13 gene sequence). Dosages used were 1, 2, 2.5, 5 or 10 mg/kgof individual oligonucleotide (as indicated below); when combinations ofoligonucleotides were administered, each oligonucleotide was given at adosage of 1, 5 or 10 mg/kg (total oligonucleotide dosages of 2, 10 and20 mg/kg, respectively). The survival times of the transplanted heartsand their hosts were monitored and recorded.

[0322] Results:

[0323] The mean survival time for untreated mice was 8.2±0.8 days (7, 8,8, 8, 9, 9 days). Treatment of the mice for 7 days with ISIS 1082 (SEQID NO: 125, unrelated control oligonucleotide) slightly reduced the meansurvival times to 7.1±0.7 days (5 mg/kg/day; 6, 7, 7, 7, 8, 8) or7.0±0.8 days (10 mg/kg/day; 6, 7, 7, 8). Treatment of the mice for sevendays with the murine B7-2 oligonucleotide ISIS 11696 (SEQ ID NO: 108)increased the mean survival time to 9.3 days at two doses (2 mg/kg/day,9.3±0.6 days, 9, 9, 10; 10 mg/kg/day, 9.3±1.3 days, 8, 9, 9, 11).Treatment of mice for seven days with an ICAM-1 oligonucleotide, ISIS3082, also increased the mean survival of the mice over several dosesSpecifically, at 1 mg/kg/day, the mean survival time (MSD) was 11.0±0.0(11, 11, 11); at 2.5 mg/kg/day, the MSD was 12.0±2.7 (10, 12, 13, 16);at 5 mg/kg/day, the MSD was 14.1±2.7 (10, 12, 12, 13, 16, 16, 17, 17);and, at 10 mg/kg/day, the MSD was 15.3±5.8 (12, 12, 13, 24). Somesynergistic effect was seen when the mice were treated for seven dayswith 1 mg/kg/day each of ISIS 3082 and 11696: the MSD was 13.8±1.0 (13,13, 14, 15)

Example 11 Detection of Nucleic Acids Encoding B7 Proteins

[0324] Oligonucleotides are radiolabeled after synthesis by ³²P-labelingat the 5′ end with polynucleotide kinase. Sambrook et al., “MolecularCloning. A Laboratory Manual,” Cold Spring Harbor Laboratory Press,1989, Volume 2, pg. 11.31. Radiolabeled oligonucleotide capable ofhybridizing to a nucleic acid encoding a B7 protein is contacted with atissue or cell sample suspected of B7 protein expression underconditions in which specific hybridization can occur, and the sample iswashed to remove unbound oligonucleotide. A similar control ismaintained wherein the radiolabeled oligonucleotide is contacted with anormal tissue or cell sample under conditions that allow specifichybridization, and the sample is washed to remove unboundoligonucleotide. Radioactivity remaining in the samples indicates boundoligonucleotide and is quantitated using a scintillation counter orother routine means. A greater amount of radioactivity remaining in thesamples, as compared to control tissues or cells, indicates increasedexpression of a B7 gene, whereas a lesser amount of radioactivity in thesamples relative to the controls indicates decreased expression of a B7gene.

[0325] Radiolabeled oligonucleotides of the invention are also useful inautoradiography. A section of tissues suspected of expressing a B7 geneis treated with radiolabeled oligonucleotide and washed as describedabove, then exposed to photographic emulsion according to standardautoradiography procedures. A control of a normal tissue section is alsomaintained. The emulsion, when developed, yields an image of silvergrains over the regions expressing a B7 gene, which is quantitated. Theextent of B7 expression is determined by comparison of the silver grainsobserved with control and test samples.

[0326] Analogous assays for fluorescent detection of expression of a B7gene use oligonucleotides of the invention which are labeled withfluorescein or other fluorescent tags. Labeled oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems, FosterCity, Calif.) using standard phosphoramidite chemistry.b-Cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidites arepurchased from Glen Research (Sterling, Va.). Incubation ofoligonucleotide and biological sample is carried out as described abovefor radiolabeled oligonucleotides except that, instead of ascintillation counter, a fluorescence microscope is used to detect thefluorescence. A greater amount of fluorescence in the: samples, ascompared to control tissues or cells, indicates increased expression ofa B7 gene, whereas a lesser amount of fluorescence in the samplesrelative to the controls indicates decreased expression of a B7 gene.

Example 12 Chimeric (Deoxy Gapped) Human B7-1 Antisense Oligonucleotides

[0327] Additional oligonucleotides targeting human B7-1 weresynthesized. Oligonucleotides were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having regions of five2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central regionof ten deoxynucleotides. Oligonucleotide sequences are shown in Table 6.

[0328] Oligonucleotides were screened as described in Example 4. Resultsare shown in Table 7.

[0329] Oligonucleotides 22315 (SEQ ID NO: 128), 22316 (SEQ ID NO: 26),22317 (SEQ ID NO: 129), 22320 (SEQ ID NO: 132), 22324 (SEQ ID NO: 135),22325 (SEQ ID NO: 136), 22334 (SEQ ID NO: 145), 22335 (SEQ ID NO: 146),22337 (SEQ ID NO: 148), and 22338 (SEQ ID NO: 36) resulted in 50% orgreater inhibition of B7-1 mRNA in this assay. TABLE 6 Nucleotide^(S)equences of Human B7-1 Chimeric (deoxy gapped) OligodeoxynucleotidesTARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGETNO. (5′−>3′) NO: ORDINATES² REGION 22313 AGACTCCACTTCTGAGATGT 1260048-0067 5′-UTR 22314 TGAAGAAAAATTCCACTTTT 127 0094-0113 5′-UTR 22315TTTAGTTTCACAGCTTGCTG 128 0112-0129 5′-UTR 22316 GCTCACGTAGAAGACCCTCC 260193-0212 5′-UTR 22317 TCCCAGGTGCAAAACAGGCA 129 0233-0252 5′-UTR 22318GTGAAAGCCAACAATTTGGA 130 0274-0293 5′-UTR 22319 CATGGCTTCAGATGCTTAGG 1310301-0320 AUG 22320 TTGAGGTATGGACACTTGGA 132 0351-0370 coding 22321GACCAGCCAGCACCAAGAGC 31 0380-0399 coding 22322 GCGTTGCCACTTCTTTCACT 1330440-0459 coding 22323 TTTTGCCAGTAGATGCGAGT 134 0501-0520 coding 22324GGCCATATATTCATGTCCCC 135 0552-0571 coding 22325 GCCAGGATCACAATGGAGAG 1360612-0631 coding 22326 GTATGTGCCCTCGTCAGATG 137 0640-0659 coding 22327TTCAGCCAGGTGTTCCCGCT 138 0697-0716 coding 22328 GGAAGTCAGCTTTGACTGAT 1390725-0744 coding 22329 CCTCCAGAGGTTGAGCAAAT 140 0798-0817 coding 22330CCAACCAGGAGAGGTGAGGC 141 0827-0846 coding 22331 GAAGCTGTGGTTGGTTGTCA 1420940-0959 coding 22332 TTGAAGGTCTGATTCACTCT 143 0987-1006 coding 22333AAGGTAATGGCCCAGGATGG 144 1050-1069 coding 22334 AAGCAGTAGGTCAGGCAGCA 1451098-1117 coding 22335 CCTTGCTTCTGCGGACACTG 146 1185-1204 coding 22336AGCCCCTTGCTTCTGCGGAC 147 1189-1208 coding 22337 TGACGGAGGCTACCTTCAGA 1481216-1235 coding 22338 GCCTCATGATCCCCACGATC 36 1254-1273 coding 22339GTAAAACAGCTTAAATTTGT 149 1286-1305 3′-UTR 22340 AGAAGAGGTTACATTAAGCA 1501398-1417 3′-UTR 22341 AGATAATGAATTGGCTGACA 151 1454-1473 3′-UTR 24733GCGTCATCATCCGCACCATC 152 control 24734 CGTTGCTTGTGCCGACAGTG 153 control24735 GCTCACGAAGAACACCTTCC 154 control

[0330] TABLE 7 Inhibition of Human B7-1 mRNA Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENE ISIS IDTARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal — —100%  — 13805 30 AUG 46% 54% 13812 36 3′-UTR 22% 78% 22313 126 5′-UTR75% 25% 22314 127 5′-UTR 69% 31% 22315 128 5′-UTR 49% 51% 22316 265′-UTR 42% 58% 22317 129 5′-UTR 43% 57% 22318 130 5′-UTR 63% 37% 22319131 AUG 68% 32% 22320 132 coding 45% 55% 22321 31 coding 57% 43% 22324135 coding 46% 54% 22325 136 coding 46% 54% 22326 137 coding 62% 38%22328 139 coding 64% 36% 22329 140 coding 59% 41% 22330 141 coding 54%46% 22331 142 coding 62% 38% 22332 143 coding 67% 33% 22333 144 coding73% 27% 22334 145 coding 43% 57% 22335 146 3′-UTR 43% 57% 22336 1473′-UTR 55% 45% 22337 148 3′-UTR 42% 58% 22338 36 3′-UTR 40% 60% 22339149 3′-UTR 69% 31% 22340 150 3′-UTR 71% 29% 22341 151 3′-UTR 59% 41%

[0331] Dose response experiments were performed on several of the moreactive oligonucleotides. The oligonucleotides were screened as describedin Example 4 except that the concentration of oligonucleotide was variedas shown in Table 8. Mismatch control oligonucleotides were included.Results are shown in Table 8.

[0332] All antisense oligonucleotides tested showed a dose responseeffect with inhibition of mRNA approximately 60% or greater. TABLE 8Dose Response of COS-7 Cells to B7-1 Chimeric (deoxy gapped) AntisenseOligonucleotides ISIS SEQ ID ASO Gene % mRNA % mRNA # NO: Target DoseExpression Inhibition basal — — — 100%  — 22316 26 5′-UTR  10 nM 99%  1%″ ″ ″  30 nM 73% 27% ″ ″ ″ 100 nM 58% 42% ″ ″ ″ 300 nM 33% 67% 24735 154control  10 nM 100%  — ″ ″ ″  30 nM 95%  5% ″ ″ ″ 100 nM 81% 19% ″ ″ ″300 nM 75% 25% 22335 146 3′-UTR  10 nM 81% 19% ″ ″ ″  30 nM 63% 37% ″ ″″ 100 nM 43% 57% ″ ″ ″ 300 nM 35% 65% 24734 153 control  10 nM 94%  6% ″″ ″  30 nM 96%  4% ″ ″ ″ 100 nM 94%  6% ″ ″ ″ 300 nM 84% 16% 22338  363′-UTR  10 nM 68% 32% ″ ″ ″  30 nM 60% 40% ″ ″ ″ 100 nM 53% 47% ″ ″ ″300 nM 41% 59% 24733 152 control  10 nM 90% 10% ″ ″ ″  30 nM 91%  9% ″ ″″ 100 nM 90% 10% ″ ″ ″ 300 nM 80% 20%

Example 13 Chimeric (Deoxy Gapped) Mouse B7-1 Antisense Oligonucleotides

[0333] Additional oligonucleotides targeting mouse B7-1 weresynthesized. Oligonucleotides were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having regions of five2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central regionof ten deoxynucleotides. Oligonucleotide sequences are shown in Table 9.

[0334] Oligonucleotides were screened as described in Example 4. Resultsare shown in Table 10. Oligonucleotides 18105 (SEQ ID NO: 156), 18106(SEQ ID NO: 157), 18109 (SEQ ID NO: 160), 18110 (SEQ ID NO: 161), 18111(SEQ ID NO: 162), 18112 (SEQ ID NO: 163), 18113 (SEQ ID NO: 164), 18114(SEQ ID NO: 165), 18115. (SEQ ID NO: 166), 1.8117 (SEQ ID NO: 168),18118 (SEQ ID NO: 169), 18119 (SEQ ID NO: 170), 18120 (SEQ ID NO: 171),18122 (SEQ ID NO: 173), and 18123 (SEQ ID NO: 174) resulted in greaterthan approximately 50% inhibition of B7-1 mRNA in this assay. TABLE 9Nucleotide Sequences of Mouse B7-1 Chimeric (deoxy gapped)Oligodeoxynucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDESEQUENCE¹ ID CO- TARGET NO. (5′−>3′) NO: ORDINATES² REGION 18104AGAGAAACTAGTAAGAGTCT 155 0018-0037 5′-UTR 18105 TGGCATCCACCCGGCAGATG 1560110-0129 5′-UTR 18106 TCGAGAAACAGAGATGTAGA 157 0144-0163 5′-UTR 18107TGGAGCTTAGGCACCTCCTA 158 0176-0195 5′-UTR 18108 TGGGGAAAGCCAGGAATCTA 1590203-0222 5′-UTR 18109 CAGCACAAAGAGAAGAATGA 160 0310-0329 coding 18110ATGAGGAGAGTTGTAACGGC 161 0409-0428 coding 18111 AAGTCCGGTTCTTATACTCG 1620515-0534 coding 18112 GCAGGTAATCCTTTTAGTGT 163 0724-0743 coding 18113GTGAAGTCCTCTGACACGTG 1640 927-0946 coding 18114 CGAATCCTGCCCCAAAGAGC 1650995-1014 coding 18115 ACTGCGCCGAATCCTGCCCC 166 1002-1021 coding 18116TTGATGATGACAACGATGAC 167 1035-1054 coding 18117 CTGTTGTTTGTTTCTCTGCT 1681098-1117 coding 18118 TGTTCAGCTAATGCTTCTTC 169 1134-1153 coding 18119GTTAACTCTATCTTGTGTCA 170 1263-1282 3′-UTR 18120 TCCACTTCAGTCATCAAGCA 1711355-1374 3′-UTR 18121 TGCTCAATACTCTCTTTTTA 172 1680-1699 3′-UTR 18122AGGCCCAGCAAACTTGCCCG 173 1330-1349 3′-UTR 18123 AACGGCAAGGCAGCAATACC 1740395-0414 coding

[0335] TABLE 10 Inhibition of Mouse B7-1 mRNA Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENE ISIS IDTARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal — —100.0% — 18104 155 5′-UTR 60.0% 40.0% 18105 156 5′-UTR 32.0% 68.0% 18106157 5′-UTR 51.0% 49.0% 18107 158 5′-UTR 58.0% 42.0% 18108 159 5′-UTR82.0% 18.0% 18109 160 coding 45.5% 54.5% 18110 161 coding 21.0% 79.0%18111 162 coding 38.0% 62.0% 18112 163 coding 42.0% 58.0% 18113 164coding 24.6% 75.4% 18114 165 coding 25.6% 74.4% 18115 166 coding 33.5%66.5% 18116 167 coding 65.6% 34.4% 18117 168 coding 46.7% 53.3% 18118169 coding 31.7% 68.3% 18119 170 3′-UTR 24.0% 76.0% 18120 171 3′-UTR25.7% 73.3% 18121 172 3′-UTR 114.0% — 18122 173 3′-UTR 42.0% 58.0% 18123174 coding 42.0% 58.0%

Example 14 Chimeric (Deoxy Gapped) Human B7-2 Antisense Oligonucleotides

[0336] Additional oligonucleotides targeting human B7-2 weresynthesized. Oligonucleotides were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having regions of five2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central regionof ten deoxynucleotides. Oligonucleotide sequences are shown in Table11.

[0337] Oligonucleotides were screened as described in Example 4. Resultsare shown in Table 12. Oligonucleotides 22284 (SEQ ID NO: 16), 22286(SEQ ID NO: 176), 22287 (SEQ ID NO: 177), 22288 (SEQ ID NO: 178), 22289(SEQ ID NO: 179), 22290 (SEQ ID NO: 180), 22291 (SEQ ID NO: 181), 22292(SEQ ID NO: 182), 22293 (SEQ ID NO: 183), 22294 (SEQ ID NO: 184), 22296(SEQ ID NO: 186), 22299 (SEQ ID NO: 189), 22300 (SEQ ID NO: 190), 22301(SEQ ID NO: 191), 22302 (SEQ ID NO: 192), 22303 (SEQ ID NO: 193), 22304(SEQ ID NO: 194), 22306 (SEQ ID NO: 196), 22307 (SEQ ID NO: 197), 22308(SEQ ID NO: 198), 22309 (SEQ ID NO: 199), 22310 (SEQ ID NO: 200), and22311 (SEQ ID NO: 201) resulted in greater than 50% inhibition of B7-2mRNA in this assay. TABLE 11 Nucleotide Sequences of Human B7-2 Chimeric(deoxy gapped) Oligodeoxynucleotides TARGET GENE SEQ NUCLEOTIDE GENEISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′−>3′) NO: ORDINATES²REGION 22284 TGCGAGCTCCCCGTACCTCC 16 0011-0030 5′-UTR 22285CAGAAGCAAGGTGGTAAGAA 175 0049-0068 5′-UTR 22286 GCCTGTCCACTGTAGCTCCA 1760113-0132 5′-UTR 22287 AGAATGTTACTCAGTCCCAT 177 0148-0167 AUG 22288TCAGAGGAGCAGCACCAGAG 178 0189-0208 coding 22289 TGGCATGGCAGGTCTGCAGT 1790232-0251 coding 22290 AGCTCACTCAGGCTTTGGTT 180 0268-0287 coding 22291TGCCTAAGTATACCTCATTC 181 0324-0343 coding 22292 CTGTCAAATTTCTCTTTGCC 1820340-0359 coding 22293 CATATACTTGGAATGAACAC 183 0359-0378 coding 22294GGTCCAACTGTCCGAATCAA 184 0392-0411 coding 22295 TGATCTGAAGATTGTGAAGT 1850417-0436 coding 22296 AAGCCCTTGTCCTTGATCTG 186 0430-0449 coding 22297TGTGATGGATGATACATTGA 187 0453-0472 coding 22298 TCAGGTTGACTGAAGTTAGC 1880529-0548 coding 22299 GTGTATAGATGAGCAGGTCA 189 0593-0612 coding 22300TCTGTGACATTATCTTGAGA 190 0694-0713 coding 22301 AAGATAAAAGCCGCGTCTTG 1910798-0817 coding 22302 AGAAAACCATCACACATATA 192 0900-0919 coding 22303AGAGTTGCGAGGCCGCTTCT 193 0947-0968 coding 22304 TCCCTCTCCATTGTGTTGGT 1940979-0998 coding 22305 CATCAGATCTTTCAGGTATA 195 1035-1054 coding 22306GGCTTTACTCTTTAATTAAA 196 1115-1134 stop 22307 GAAATCAAAAAGGTTGCCCA 1971178-1197 3′-UTR 22308 GGAGTCCTGGAGCCCCCTTA 198 1231-1250 3′-UTR 22309TTGGCATACGGAGCAGAGCT 199 1281-1300 3′-UTR 22310 TGTGCTCTGAAGTGAAAAGA 2001327-1346 3′-UTR 22311 GGCTTGGCCCATAAGTGTGC 201 1342-1361 3′-UTR 22312CCTAAATTTTATTTCCAGGT 202 1379-1398 3′-UTR 24736 GCTCCAAGTGTCCCAATGAA 203control 24737 AGTATGTTTCTCACTCCGAT 204 control 24738TGCCAGCACCCGGTACGTCC 205 control

[0338] TABLE 12 Inhibition of Human B7-2 mRNA Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENE ISIS IDTARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal — —100%   0% 10373  16 5′-UTR 24% 76% 22284  16 5′-UTR 30% 70% 22285 1755′-UTR 74% 26% 22286 176 5′-UTR 39% 61% 22287 177 AUG 27% 73% 22288 178coding 38% 62% 22289 179 coding 41% 59% 22290 180 coding 42% 58% 22291181 coding 41% 59% 22292 182 coding 39% 61% 22293 183 coding 43% 57%22294 184 coding 21% 79% 22295 185 coding 66% 34% 22296 186 coding 42%58% 22297 187 coding 54% 46% 22298 188 coding 53% 47% 22299 189 coding46% 54% 22300 190 coding 39% 61% 22301 191 coding 51% 49% 22302 192coding 41% 59% 22303 193 coding 46% 54% 22304 194 coding 41% 59% 22305195 coding 57% 43% 22306 196 stop 44% 56% 22307 197 3′-UTR 45% 55% 22308198 3′-UTR 40% 60% 22309 199 3′-UTR 42% 58% 22310 200 3′-UTR 41% 59%22311 201 3′-UTR 49% 51% 22312 202 3′-UTR 83% 17%

[0339] Dose response experiments were performed on several of the moreactive oligonucleotides. The oligonucleotides were screened as describedin Example 4 except that the concentration of oligonucleotide was variedas shown in Table 13. Mismatch control oligonucleotides were included.Results are shown in Table 13.

[0340] All antisense oligonucleotides tested showed a dose responseeffect with maximum inhibition of mRNA approximately 50% or greater.TABLE 13 Dose Response of COS-7 Cells to B7-2 Chimeric (deoxy gapped)Antisense Oligonucleotides ISIS SEQ ID ASO Gene % mRNA % mRNA # NO:Target Dose Expression Inhibition basal — — — 100%  — 22284 16 5′-UTR 10 nM 92%  8% ″ ″ ″  30 nM 72% 28% ″ ″ ″ 100 nM 59% 41% ″ ″ ″ 300 nM48% 52% 24738 205 control  10 nM 81% 19% ″ ″ ″  30 nM 92%  8% ″ ″ ″ 100nM 101% — ″ ″ ″ 300 nM 124% — 22287 177 AUG  10 nM 93%  7% ″ ″ ″  30 nM79% 21% ″ ″ ″ 100 nM 66% 34% ″ ″ ″ 300 nM 45% 55% 24737 204 control  10nM 85% 15% ″ ″ ″  30 nM 95%  5% ″ ″ ″ 100 nM 87% 13% ″ ″ ″ 300 nM 99% 1% 22294 184 coding  10 nM 93%  7% ″ ″ ″  30 nM 95%  5% ″ ″ ″ 100 nM58% 42% ″ ″ ″ 300 nM 45% 55% 24736 203 control  10 nM 102%  — ″ ″ ″  30nM 101%  — ″ ″ ″ 100 nM 100%  — ″ ″ ″ 300 nM 107%  —

Example 15 Chimeric (deoxy gapped) Mouse B7-2 Antisense Oligonucleotides

[0341] Additional oligonucleotides targeting mouse B7-2 weresynthesized. Oligonucleotides were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having regions of five2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central regionof ten deoxynucleotides. Oligonucleotide sequences are shown in Table14.

[0342] Oligonucleotides were screened as described in Example 4. Resultsare shown in Table 15.

[0343] Oligonucleotides 18084 (SEQ ID NO: 206), 18085 (SEQ ID NO: 207),18086 (SEQ ID NO: 208), 18087 (SEQ ID NO: 209), 18089 (SEQ ID NO: 211),18090 (SEQ ID NO: 212), 18091 (SEQ ID NO: 213), 18093 (SEQ ID NO: 215),18095 (SEQ ID NO: 217), 18096 (SEQ ID NO: 218), 18097 (SEQ ID NO: 219),18098 (SEQ ID NO: 108), 18102 (SEQ ID NO: 223), and 18103 (SEQ ID NO:224) resulted in 50% or greater inhibition of B7-2 mRNA expression inthis assay. TABLE 14 Nucleotide Sequences of Mouse B7-2 Chimeric (deoxygapped) oligodeoxynucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISISNUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′−>3′) NO: ORDINATES² REGION18084 GCTGCCTACAGGAGCCACTC 206 0003-0022 5′-UTR 18085TCAAGTCCGTGCTGCCTACA 207 0013-0032 5′-UTR 18086 GTCTACAGGAGTCTGGTTGT 2080033-0052 5′-UTR 18087 AGCTTGCGTCTCCACGGAAA 209 0152-0171 coding 18088TCACACTATCAAGTTTCTCT 210 0297-0316 coding 18089 GTCAAAGCTCGTGCGGCCCA 2110329-0348 coding 18090 GTGAAGTCGTACAGTCCAGT 212 0356-0375 coding 18091GTGACCTTGCTTAGACGTGC 213 0551-0570 coding 18092 CATCTTCTTAGGTTTCGGGT 2140569-0588 coding 18093 GGCTGTTGGAGATACTGAAC 215 0663-0682 coding 18094GGGAATGAAAGAGAGAGGCT 216 0679-0698 coding 18095 ACATACAATGATGAGCAGCA 2170854-0873 coding 18096 GTCTCTCTGTCAGCGTTACT 218 0934-0953 coding 18097TGCCAAGCCCATGGTGCATC 219 0092-0111 AUG 18098 GGATTGCCAAGCCCATGGTG 1080096-0115 AUG 18099 GCAATTTGGGGTTCAAGTTC 220 0967-0986 coding 18100CAATCAGCTGAGAACATTTT 221 1087-1106 3′-UTR 18101 TTTTGTATAAAACAATCATA 2220403-0422 coding 18102 CCTTCACTCTGCATTTGGTT 223 0995-1014 stop 18103TGCATGTTATCACCATACTC 224 0616-0635 coding

[0344] TABLE 15 Inhibition of Mouse B7-2 mRNA Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENE ISIS IDTARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal — —100.0% 0.0% 18084 206 5′-UTR 36.4% 63.6% 18085 207 5′-UTR 35.0% 65.0%18086 208 5′-UTR 40.1% 59.9% 18087 209 coding 42.1% 57.9% 18088 210coding 52.3% 47.7% 18089 211 coding 20.9% 79.1% 18090 212 coding 36.6%63.4% 18091 213 coding 37.1% 62.9% 18092 214 coding 58.9% 41.1% 18093215 coding 32.7% 67.3% 18094 216 coding 63.8% 36.2% 18095 217 coding34.3% 65.7% 18096 218 coding 32.3% 67.7% 18097 219 AUG 24.5% 75.5% 18098108 AUG 32.2% 67.8% 18099 220 coding 66.8% 33.2% 18100 221 3′-UTR 67.2%32.8% 18101 222 coding 88.9% 11.1% 18102 223 stop 33.8% 66.2% 18103 224coding 30.2% 69.8%

Example 16 Effect of B7 Antisense Oligonucleotides on Cell SurfaceExpression

[0345] B7 antisense oligonucleotides were tested for their effect oncell surface expression of both B7-1 and B7-2. Cell surface expressionwas measured as described in Example 2. Experiments were done for bothhuman B7 and mouse B7. Results for human B7 are shown in Table 16.Results for mouse B7 are shown in Table 17.

[0346] In both species, B7-1 antisense oligonucleotides were able tospecifically reduce the cell surface expression of B7-1. B7-2 antisenseoligonucleotides were specific for the B7-2 family member. Theseoligonucleotides were also specific for their effect on B7-1 and B7-2mRNA levels. TABLE 16 Inhibition of Human B7 Cell Surface Expression byChimeric (deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ ISISID GENE % B7-1 % B7-2 No: NO: TARGET EXPRESSION EXPRESSION basal — —100%   0% 22316 26 B7-1 31% 100% 22317 129 B7-1 28%  91% 22320 132 B7-137%  86% 22324 135 B7-1 37%  91% 22325 136 B7-1 32%  89% 22334 145 B7-128%  92% 22335 146 B7-1 23%  95% 22337 148 B7-1 48% 101% 22338 36 B7-122%  96% 22284 16 B7-2 88%  32% 22287 177 B7-2 92%  35% 22294 184 B7-277%  28%

[0347] TABLE 17 Inhibition of Mouse B7 Cell Surface Expression byChimeric (deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENEISIS ID TARGET % B7-1 % B7-2 No: NO: REGION EXPRESSION EXPRESSION basal— — 100%   0% 18089 211 B7-2 85% 36% 18097 219 B7-2 87% 28% 18110 161B7-1 31% 93% 18113 164 B7-1 25% 91% 18119 170 B7-1 27% 98%

[0348] Dose response experiments were performed on several of the moreactive human B7-1 antisense oligonucleotides. The oligonucleotides werescreened as described in Example 2 except that the concentration ofoligonucleotide was varied as shown in Table 18. Results are shown Table18.

[0349] All antisense oligonucleotides tested showed a dose responseeffect with inhibition of cell surface expression approximately 60% orgreater. TABLE 18 Dose Response of COS-7 Cells to B7-1 Chimeric (deoxygapped) Antisense Oligonucleotides ISIS SEQ ID ASO Gene % Surface %Surface # NO: Target Dose Expression Inhibition basal — — — 100%  —22316 26 5′-UTR  10 nM 74% 26% ″ ″ ″  30 nM 74% 26% ″ ″ ″ 100 nM 47% 53%″ ″ ″ 300 nM 34% 66% 22335 146  3′-UTR  10 nM 81% 19% ″ ″ ″  30 nM 69%31% ″ ″ ″ 100 nM 47% 53% ″ ″ ″ 300 nM 38% 62% 22338 36 3′-UTR  10 nM 78%22% ″ ″ ″  30 nM 65% 35% ″ ″ ″ 100 nM 50% 50% ″ ″ ″ 300 nM 40% 60%

[0350] Dose response experiments were performed on several of the moreactive human B7-2 antisense oligonucleotides. The oligonucleotides werescreened as described in Example 2 except that the concentration ofoligonucleotide was varied as shown in Table 19. Results are shown inTable 19.

[0351] All antisense oligonucleotides tested showed a dose responseeffect with maximum inhibition of cell surface expression 85% orgreater. TABLE 19 Dose Response of COS-7 Cells to B7-2 Chimeric (deoxygapped) Antisense Oligonucleotides ISIS SEQ ID ASO Gene % Surface %Surface # NO: Target Dose Expression Inhibition basal — — — 100%  —22284  16 5′-UTR  10 nM 63% 37% ″ ″ ″  30 nM 60% 40% ″ ″ ″ 100 nM 37%63% ″ ″ ″ 300 nM 15% 85% 22287 177 AUG  10 nM 93%  7% ″ ″ ″  30 nM 60%40% ″ ″ ″ 100 nM 32% 68% ″ ″ ″ 300 nM 15% 85% 22294 184 coding  10 nM89% 11% ″ ″ ″  30 nM 62% 38% ″ ″ ″ 100 nM 29% 71% ″ ″ ″ 300 nM 12% 88%

Example 17 Effect of B7-1 Antisense Oligonucleotides in a Murine Modelfor Rheumatoid Arthritis

[0352] Collagen-induced arthritis (CIA) was used as a murine model forarthritis (Mussener, A., et al., Clin. Exp. Immunol., 1997, 107,485-493). Female DBA/1LacJ mice (Jackson Laboratories, Bar Harbor, Me.)between the ages of 6 and 8 weeks were used to assess the activity ofB7-1 antisense oligonucleotides.

[0353] On day 0, the mice were immunized at the base of the tail with100 μg of bovine type II collagen which is emulsified in CompleteFreund's Adjuvant (CFA). On day 7, a second booster dose of collagen wasadministered by the same route. On day 14, the mice were injectedsubcutaneously with 100 μg of LPS. Oligonucleotide was administeredintraperitoneally daily (10 mg/kg bolus) starting on day −3 (three daysbefore day 0) and continuing for the duration of the study.Oligonucleotide 17456 (SEQ ID NO. 173) is a fully phosphorothioatedanalog of 18122.

[0354] Weights were recorded weekly. Mice were inspected daily for theonset of CIA. Paw widths are rear ankle widths of affected andunaffected joints were measured three times a week using a constanttension caliper. Limbs were clinically evaluated and graded on a scalefrom 0-4 (with 4 being the highest).

[0355] Results are shown in Table 20. Treatment with B7-1 and B7-2antisense oligonucleotides was able to reduce the incidence of thedisease, but had modest effects on severity. The combination of 17456(SEQ ID NO. 173) and 11696 (SEQ ID NO. 108) was able to significantlyreduce the incidence of the disease and its severity. TABLE 20 Effect ofB7 antisense oligonucleotide on CIA SEQ Dose % ISIS #(s) ID NO mg/kgIncidence Peak day¹ Severity² control — 70% 6.7 ± 2.9 3.2 ± 1.1 17456(B7-1) 173 10 50% 12.1 ± 4.6  2.7 ± 1.3 11696 (B7-2) 108 10 37.5%   11.6± 4.5  3.4 ± 1.8 17456/11696 10 30% 1.0 ± 0.6 0.7 ± 0.4 18110 (B7-1 16110 55.6%   2.0 ± 0.8 2.0 ± 1.3 18089 (B7-2 211 10 44.4%   6.8 ± 2.2 2.3± 1.3 18110/18089 10 60% 11.6 ± 0.7  4.5 ± 1.7

Example 18 Effect of B7-1 Antisense Oligonucleotides in a Murine Modelfor Multiple Sclerosis

[0356] Experimental autoimmune encephalomyelitis (EAE) is a commonlyaccepted murine model for multiple sclerosis (Myers, K. J., et al., J.Neuroimmunol., 1992, 41, 1-8). SJL/H, PL/J, (SJLxPL/J)F1, (SJLxBalb/c)F1and Balb/c female mice between the ages of 6 and 12 weeks are used totest the activity of a B7-1 antisense oligonucleotide.

[0357] The mice are immunized in the two rear foot pads and base of thetail with an emulsion consisting of encephalitogenic protein or peptide(according to Myers, K. J., et al., J. of Immunol., 1993, 151,2252-2260) in Complete Freund's Adjuvant supplemented with heat killedMycobacterium tuberculosis. Two days later, the mice receive anintravenous injection of 500 ng Bordetella pertussis toxin andadditional adjuvant.

[0358] Alternatively, the disease may also be induced by the adoptivetransfer of T-cells. T-cells are obtained from the draining of the lymphnodes of mice immunized with encephalitogenic protein or peptide in CFA.The T cells are grown in tissue culture for several days and theninjected intravenously into naive syngeneic recipients.

[0359] Mice are monitored and scored daily on a 0-5 scale for signals ofthe disease, including loss of tail muscle tone., wobbly gait, andvarious degrees of paralysis.

[0360] Oligonucleotide 17456 (SEQ ID NO. 173), a fully phosphorothioatedanalog of 18122, was compared to a saline control and a fullyphosphorothioated oligonucleotide of random sequence (Oligonucleotide17460). Results of this experiment are shown in FIG. 11.

[0361] As shown in FIG. 11, for all doses of oligonucleotide 17456tested, there is a protective effect, i.e. a reduction of diseaseseverity. At 0.2 mg/kg, this protective effect is greatly reduced afterday 20, but at the higher doses tested, the protective effect remainsthroughout the course of the experiment (day 40). The controloligonucleotide gave results similar to that obtained with the salinecontrol.

Example 19 Additional Antisense Oligonucleotides Targeted to Human B7-1

[0362] Additional oligonucleotides targeting human B7-1 weresynthesized. Oligonucleotides were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having regions of five2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central regionof ten deoxynucleotides. Oligonucleotide sequences are shown in Table21.

[0363] The human promonocytic leukaemia cell line, THP-1 (American TypeCulture Collection, Manassas, Va.) was maintained in RPMI 1640 growthmedia supplemented with 10% fetal calf serum (FCS; Life Technologies,Rockville, Md.). A total of 1×10 cells were electroporated at anoligonucleotide concentration of 10 micromolar in 2 mm cuvettes, usingan Electrocell Manipulator 600 instrument (Biotechnologies andExperimental Research, Inc.) employing 200 V, 1000 μF. Electroporatedcells were then transferred to petri dishes and allowed to recover for16 hrs. Cells were then induced with LPS at a final concentration of 1μg/ml for 16 hours. RNA was isolated and processed as described inprevious examples. Results are shown in Table 22.

[0364] Oligonucleotides 113492, 113495, 113498, 113499, 113501, 113502,113504, 113505, 113507, 113510, 113511, 113513 and 113514 (SEQ ID NO:228, 231, 234, 235, 237, 238, 240, 241, 243, 246, 247, 249 and 250)resulted in 50% or greater inhibition of B7-1 mRNA expression in thisassay. TABLE 21 Nucleotide Sequences of Human B7-1 Chimeric (deoxygapped) Oligodeoxynucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISISNUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′−>3′) NO. ORDINATES² REGION113489 CCCTCCAGTGATGTTTACAA 225 179 5′ UTR 113490 GAAGACCCTCCAGTGATGTT226 184 5′ UTR 113491 CGTAGAAGACCCTCCAGTGA 227 188 5′ UTR 113492TTCCCAGGTGCAAAACAGGC 228 234 5′ UTR 113493 TGCCTTCAGATGCTTAGGGT 229 2995′ UTR 113494 CCTCCGTGTGTGGCCCATGG 230 316 AUG 113495GGTGATGTTCCCTGCCTCCG 231 330 Coding 113496 GATCGTGATGTTCCCTGCCT 232 333Coding 113497 AGGTATGGACACTTGGATGG 233 348 Coding 113498GAAAGACCAGCCAGCACCAA 234 384 Coding 113499 CAGCGTTGCCACTTCTTTCA 235 442Coding 113500 GTGACCACAGGACAGCGTTG 236 454 Coding 113501AGATGCGAGTTTGTGCCAGC 237 491 Coding 113502 CCTTTTGCCAGTAGATGCGA 238 503Coding 113503 CGGTTCTTGTACTCGGGCCA 239 567 Coding 11350CGCAGAGCCAGGATCACAAT 240 618 Coding 113505 CTTCAGCCAGGTGTTCCCGC 241 698Coding 113506 TAACGTCACTTCAGCCAGGT 242 706 Coding 113507TTCTCCATTTTCCAACCAGG 243 838 Coding 113508 CTGTTGTGTTGATGGCATTT 244 863Coding 113509 CATGAAGCTGTGGTTGGTTG 245 943 Coding 113510AGGAAAATGCTCTTGCTTGG 246 1018 Coding 113511 TGGGAGCAGGTTATCAGGAA 2471033 Coding 113512 TAAGGTAATGGCCCAGGATG 248 1051 Coding 113513GGTCAGGCAGCATATCACAA 249 1090 Coding 113514 GCCCCTTGCTTCTGCGGACA 2501188 3′ UTR 113515 AGATCTTTTCAGCCCCTTGC 251 1199 3′ UTR 113516TTTGTTAAGGGAAGAATGCC 252 1271 3′ UTR 113517 AAACGAGAGGGATGCCAGCC 2531362 3′ UTR 113518 CAAGACAATTCAAGATGGCA 254 1436 3′ UTR

[0365] TABLE 22 Inhibition of Human B7-1 mRNA Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENE ISIS IDTARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION 113489 225 5′UTR 122 — 113490 226 5′ UTR 183 — 113491 227 5′ UTR 179 — 113492 228 5′UTR 27 73 113493 229 5′ UTR 488 — 113494 230 AUG 77 23 113495 231 Coding43 57 113496 232 Coding 71 29 113497 233 Coding 78 22 113498 234 Coding37 63 113499 235 Coding 25 75 113500 236 Coding 83 17 113501 237 Coding36 64 113502 238 Coding 26 74 113503 239 Coding 65 35 113504 240 Coding46 54 113505 241 Coding 40 60 113506 242 Coding 105 — 113507 243 Coding36 64 113508 244 Coding 117 — 113509 245 Coding 62 38 113510 246 Coding43 57 113511 247 Coding 48 52 113512 248 Coding 73 27 113513 249 Coding48 52 113514 250 3′ UTR 35 65 113515 251 3′ UTR 184 — 113516 252 3′ UTR83 17 113517 253 3′ UTR 201 — 113518 254 3′ UTR 97 03

Example 20 Additional Antisense Oligonucleotides Targeted to Human B7-2

[0366] Additional oligonucleotides targeting human B7-2 weresynthesized. Oligonucleotides were synthesized as uniformlyphosphorothioate chimeric oligonucleotides having regions of five2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central regionof ten deoxynucleotides. Oligonucleotide sequences are shown in Table23.

[0367] The human promonocytic leukaemia cell line, THP-1 (American TypeCulture Collection, Manassas, Va.) was maintained in RPMI 1640 growthmedia supplemented with 10% fetal calf serum (FCS; Life Technologies,Rockville, Md.). A total of 1×10⁷ cells were electroporated at anoligonucleotide concentration of 10 micromolar in 2 mm cuvettes, usingan Electrocell Manipulator 600 instrument (Biotechnologies andExperimental Research, Inc.) employing 200 V, 1000 μF. Electroporatedcells were then transferred to petri dishes and allowed to recover for16 hrs Cells were then induced with LPS and dibutyryl cAMP (500 μM) for16 hours. RNA was isolated and processed as described in previousexamples. Results are shown in Table 24.

[0368] Oligonucleotides ISIS 113131, 113132, 113134, 113138, 113142,113144, 113145, 113146, 113147, 113148, 113149, 113150, 113153, 113155,113157, 113158, 113159 and 113160 (SEQ ID NO: 255, 256, 258, 262, 266,268, 269, 270, 271, 272, 273, 274, 277, 279, 281, 282, 283 and 284)resulted in 50% or greater inhibition of B7-2 mRNA expression in thisassay. TABLE 23 Nucleotide Sequences of Human B7-2 Chimeric (deoxygapped) Oligodeoxynucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISISNUCLEOTIDE SEQUENCE¹ ID CO TARGET NO. (5′−>3′) NO: ORDINATES² REGION113131 CGTGTGTCTGTGCTAGTCCC 255 38 5′ UTR 113132 GCTGCTTCTGCTGTGACCTA256 83 5′ UTR 113133 TATTTGCGAGCTCCCCGTAC 257 15 5′ UTR 113134GCATAAGCACAGCAGCATTC 258 79 5′ UTR 113135 TCCAAAAAGAGACCAGATGC 259 975′ UTR 113136 AAATGCCTGTCCACTGTAGC 260 117 5′ UTR 113137CTTCAGAGGAGCAGCACCAG 261 191 Coding 113138 GAATCTTCAGAGGAGCAGCA 262 195Coding 113139 CAAATTGGCATGGCAGGTCT 263 237 coding 113140GCTTTGGTTTTGAGAGTTTG 264 257 Coding 113141 AGGCTTTGGTTTTGAGAGTT 265 259Coding 113142 GCTCACTCAGGCTTTCGTTT 266 267 Coding 113143GGTCCTGCCAAAATACTACT 267 288. Coding 113144 AGCCCTTGTCCTTGATCTGA 268 429Coding 113145 TCTGGCCTTTTTGTGATGGA 269 464 Coding 113146AATCATTCCTGTCGGCTTTT 270 473 Coding 113147 CCGTGTATACATGAGCAGGT 271 595Coding 113148 ACCGTGTATAGATGAGCAGG 272 596 Coding 113149TCATCTTCTTAGGTTCTGGG 273 618 Coding 113150 ACAAGCTGATGGAAACCTCG 274 720Coding 113151 TGCTCGTAACATCAGGGAAT 275 747 Coding 113152AAGATGGTCATATTGCTCGT 276 760 Coding 113153 CGCGTCTTGTCAGTTTCCAG 277 787Coding 113154 CAGCTGTAATCCAAGGAATG 278 864 Coding 113155GGGCTTCATCACATCTTTCA 279 1041 Coding 113156 CATGTATCACTTTTGTCGCA 2801093 Coding 113157 AGCCCCCTTATTACTCATGG 281 1221 3′ UTR 113158GGAGTTACAGGGAGGCTATT 282 1261 3′ UTR 113159 AGTCTCCTCTTGGCATACGG 2831290 3′ UTR 113160 CCCATAAGTGTGCTCTGAAG 284 1335 3′ UTR

[0369] TABLE 24 Inhibition of Human B7-2 mRNA Expression by Chimeric(deoxy gapped) Phosphorothioate Oligodeoxynucleotides SEQ GENE ISIS IDTARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION 113131 255 5′UTR 13 87 113132 256 5′ UTR 17 83 113133 257 5′ UTR 214 — 113134 258 5′UTR 27 73 113135 259 5′ UTR 66 34 113136 260 5′ UTR 81 19 113137 261Coding 57 43 113138 262 Coding 12 88 113140 264 Coding 214 — 113141 265Coding 126 — 113142 266 Coding 35 65 113143 267 Coding 118 — 113144 268Coding 41 59 113145 269 Coding 46 54 113146 270 Coding 32 68 113147 271Coding 35 65 113148 272 Coding 23 77 113149 273 Coding 29 71 113150 274Coding 19 81 113151 275 Coding 208 — 113152 276 Coding 89 11 113153 277Coding 19 81 113154 278 Coding 63 37 113155 279 Coding 13 87 113156 280Coding 83 17 113157 281 3′ UTR 13 87 113158 282 3′ UTR 20 80 113159 2833′ UTR 43 57 113160 284 3′ UTR 09 91

Example 21 Human Skin Psoriasis Model

[0370] Animal models of psoriasis based on xenotransplantation of humanskin from psoriatic patients are advantageous because they involve thedirect study of affected human tissue. Psoriasis is solely a disease ofthe skin and consequently, engraftment of human psoriatic skin onto SCIDmice allows psoriasis to be created with a high degree of fidelity inmice.

[0371] BALB/cByJSmn-Prkdcscid/J SCID mice (4-6 weeks old) of either sex(Jackson Laboratory, Bar Harbor, Me.) were maintained in a pathogen freeenvironment. At 6-8 weeks of age, mice were anesthetized byintraperitoneal injection of 30 mg/kg body weight ketamine-HCl and 1mg/kg body weight acepromazine. After anesthesia, mice were prepared fortransplantation by shaving the hair from the dorsal skin, 2 cm away fromthe head. The area was then sterilized and cleaned with povidone iodideand alcohol. Graft beds of about 1 cm×1 cm were created on the shavedareas by removing full thickness skin down to the fascia. Partialthickness human skin was then orthotopically transferred onto the graftbed. The transplants were held in place by gluing the human skin tomouse-to-mouse skin with Nexband liquid, a veterinary bandage(Veterinary Products Laboratories, Phoenix, Ariz.). Finally, thetransplant and the wounds were covered with a thick layer of antibioticointment. After 4 weeks of transplantation, a 2 mm punch biopsy wasobtained to confirm the acceptance of the graft, and the origin of theskin in the transplant area. Only mice whose grafts did not show signsof infection were used for the study. Normal human skin was obtainedfrom elective plastic surgeries and psoriatic plaques were obtained fromshave biopsies from psoriatic volunteers. Partial thickness skin wasprepared by dermatome shaving of the skin and transplanted to the mouseas described above for the psoriatic skin.

[0372] Animals (n=5) were topically treated with 2.5% (w/w) of eachantisense oligonucleotide in a cream formulation comprising 10%isopropyl mylistate, 10% glyceryl moriooleate, 3% cetostearyl alcohol,10% polyoxyl-20-cetyl ether, 6% poloxamer 407, 2.5% phenoxyethanol, 0.5%methylparaben, 0.5% propylparaben and water (final pH about 7.5).

[0373] The following oligonucleotides were used: human B7-1(5′-TTCCCAGGTGCAAAACAGGC-3′; SEQ ID NO: 228) (ISIS 113492) and humanB7-2 (5′-CGTGTGTCTGTGCTAGTCCC-3′; SEQ ID NO: 255) (ISIS 113131). Bothsequences contained only phosphorothioate linkages and had 2′-MOEmodifications at nucleotides 1-5 and 16-20.

[0374] Plaques from the same patients were also transplanted ontocontrol mice (n=5) and treated only with the vehicle of the active creampreparation. Both groups received the topical preparation twice a dayfor 4 weeks. Within 3-4 weeks the animals were sacrificed and 4 mm punchbiopsies were taken from each xenograft. Biopsies were fixed in formalinfor paraffin embedding and/or transferred to cryotubes and snap-frozenin liquid nitrogen and stored at −80° C.

[0375] Significant histological improvement marked by reduction ofhyperkeratosis, acanthosis and lymphonuclear cellular infiltrates wasobserved in mice treated with the antisense oligonucleotides. Rete pegs,finger-like projections of the epidermis into the dermis, were alsomeasured. These are phenotypic markers for psoriasis which lengthen asthe disease progresses. The shortening of these rete pegs are a goodmeasure of anti-psoriatic activity. In mice treated with the activeagent, the rete pegs changed from 238.56±98.3 μm to 168.4±96.62 μm(p<0.05), whereas in the control group the rete pegs before and aftertreatment were 279.93±40.56 μm and 294.65±45.64 μm, respectively(p>0.1). HLA-DR positive lymphocytic infiltrates and intraepidermal CD8positive lymphocytes were significantly reduced in the transplantedplaques treated with the antisense oligonucleotide cream. These resultsshow that antisense oligonucleotides to B7 inhibit psoriasis-inducedinflammation and have therapeutic efficacy in the treatment ofpsoriasis.

Example 22 Mouse Model of Allergic Inflammation

[0376] In the mouse model of allergic inflammation, mice were sensitizedand challenged with aerosolized chicken ovalbumin (OVA). Airwayresponsiveness was assessed by inducing airflow obstruction with amethacholine aerosol using a noninvasive method. This methodologyutilized unrestrained conscious mice that are placed into the mainchamber of a plthysmograph (Buxco Electronics, Inc., Troy, N.Y.).Pressure differences between this chamber and a reference chamber wereused to extrapolate minute volume, breathing frequency and enhancedpause (Penh). Penh is a dimensionless parameter that is a function oftotal pulmonary airflow in mice (i.e., the sum of the airflow in theupper and lower respiratory tracts) during the respiratory cycle of theanimal. The lower the PENH, the greater the airflow. This parameterclosely correlates with lung resistance as measured by traditionalinvasive techniques using ventilated animals (Hamelmann . . . . Gelfand,1997). Dose-response data were plotted as raw Penh values to increasingconcentrations of methacholine. This system was used to test theefficacy of antisense oligonucleotides targeted to human B7-1 and B7-2.

[0377] There are several important features common to human asthma andthe mouse model of allergic inflammation. One of these is pulmonaryinflammation, in which cytokine expression and Th2 profile is dominant.Another is goblet cell hyperplasia with increased mucus production.Lastly, airway hyperresponsiveness (AHR), occurs resulting in increasedsensitivity to cholinergic receptor agonists such as acetylcholine ormethacholine. The compositions and methods of the present invention maybe used to treat AHR and pulmonary inflammation.

[0378] Ovalbumin-Induced Allergic Inflammation

[0379] Female Balb/c mice (Charles Rivers Laboratory, Taconic Farms, NY)were maintained in micro-isolator cages housed in a specificpathogen-free (SPF) facility. The sentinel cages within the animalcolony surveyed negative for viral antibodies and the presence of knownmouse pathogens. Mice were sensitized and challenged with aerosolizedchicken OVA. Briefly, 20 μg alum-precipitated OVA was injectedintraperitoneally on days 0 and 14. On day 24, 25 and 26, the animalswere exposed for 20 minutes to 1.0% OVA (in saline) by nebulization. Thechallenge was conducted using an ultrasonic nebulizer (PulmoSonic, TheDeVilbiss Co., Somerset, PA). Animals were analyzed about 24 hoursfollowing the last nebulization using the Buxco electronics Biosystem.Lung function (Penh), lung histology (cell infiltration and mucusproduction), target mRNA reduction in the lung, inflammation (BAL celltype & number, cytokine levels), spleen weight and serum AST/ALT weredetermined.

[0380] This method has been used to show that prophylactic treatmentwith an anti-B7.2 monoclinal antibody continued throughout thesensitization and challenge periods decreases OVA-specific serum IgE andIgE levels, IL-4 and IFN-γ levels in bronchoalveolar lavage (BAL) fluid,airway eosinophilia and airway hyperresponsiveness (Haczku et al., Am.J. Respir. Crit. Care Med. 159:1638-1643, 1999). Treatment duringantigen challenge with both anti-B7.1 and anti-B7.2 mAbs is effective;however, either mAb alone is only partially active (Mathur et al.,21:498-509, 1999). However, the anti-B7.2 mAb had no activity whenadministered after the OVA challenge. The anti-B7.1 monoclonal antibodyhad no effect, either prophylactically or post-antigen challenge. Thus,there is a need for an effective B7 inhibitor which can be administeredafter antigen challenge, and which will reduce airwayhyperresponsiveness and pulmonary inflammation. As described below, theantisense oligonucleotides of the present inventors fit thisdescription.

[0381] Oligonucleotide Administration

[0382] Antisense oligonucleotides (ASOs) were dissolved in saline andused to intratracheally dose mice every day, four times per day, fromdays 15-26 of the OVA sensitization and challenge protocol.Specifically, the mice were anesthetized with isofluorane and placed ona board with the front teeth hung from a line. The nose was covered andthe animal's tongue was extended with forceps and 25 μl of various dosesof ASO, or an equivalent volume of saline (control) was placed at theback of the tongue until inhaled into the lung. The deposition patternof an ASO in the lung, ISIS 13920 (5′-TCCGTCATCGCTCCTCAGGG-3′; SEQ IDNO:285) was also examined by immunohistochemical staining using amonoclonal antibody to the oligonucleotide, and showed that the ASO istaken up; throughout the lung, most strongly by antigen presenting cells(APCs) and alveolar epithelium.

[0383] The B7 oligonucleotides used were: (ISIS 121844; SEQ ID NO: 286)B7-1: 5′-GCTCAGCCTTTCCACTTCAG-3′ (ISIS 121874; SEQ ID NO: 287) B7-2:5′-GCTCAGCCTTTCCACTTCAG-3′

[0384] Both of these oligonucleotides are phosphorothioates with 2′-MOEmodifications on nucleotides 1-5 and 16-20, and 2′-deoxy at positions6-15. These ASOs were identified by mouse-targeted ASO screening bytarget mRNA reduction in mouse cell lines. For B7-2, 19 mouse-targetedASOs were screened by target mRNA reduction (Northern analysis) in IC-21macrophages. Dose-response confirmation led to selection of ISIS 121874(>70% reduction at 25 nM). For B7-1, 22 mouse-targeted ASOs werescreened by target mRNA reduction (RT-PCR) in L-929 fibroblasts.Dose-response confirmation led to selection of ISIS 121844 (>70%reduction at 100 nM). No cross hybridization was predicted, and nocross-target reduction was detected in transfected cells.

[0385] RT-PCR Analysis

[0386] RNA was harvested from experimental lungs removed on day 28 ofthe OVA protocol. B7.2 and B7.1 levels were measured by quantitativeRT-PCR using the Applied Biosystems PRISM 7700 Sequence Detection System(Applied Biosystems, Foster City, Calif.). Primers and probes used forthese studies were synthesized by Operon Technologies (Alameda, Calif.).The primer and probe sequences were as follows:

[0387] B7-2: B7-2: forward: 5′-GGCCCTCCTCCTTGTGATG-3′ (SEQ ID NO: 288)probe: 5′-/56- (SEQ ID NO: 289)FAM/TGCTCATCATTGTATGTCACAAGAAGCCG/36-TAMTph/-3′ reverse:5′-CTGGGCCTGCTAGGCTGAT-3′ (SEQ ID NO: 290) B7-1: forward:5′-CAGCAAGCTACGGGCAAGTT-3′ (SEQ ID NO: 291) probe: 5′-/56- (SEQ ID NO:292) FAM/TGGGCCTTTGATTGCTTGATGACTGAA/36-TAMTph/-3′ reverse:5′-GTGGGCTCAGCCTTTCCA-3′ (SEQ ID NO: 293)

[0388] Collection of Bronchial Alveolar Lavage (BAL) Fluid and BloodSerum for the Determination of Cytokine and Chemokine Levels

[0389] Animals were injected with a lethal dose of ketamine, the tracheawas exposed and a cannula was inserted and secured by sutures. The lungswere lavaged twice with 0.5 ml aliquots of ice cold PBS with 0.2% FCS.

[0390] The recovered BAL fluid was centrifuged at 1,000 rpm for 10 minat 4° C., frozen on dry ice and stored at −80° C. until used. Luminexwas used to measure cytokine levels in BAL fluid and serum.

[0391] BAL Cell Counts and Differentials

[0392] Cytospins of cells recovered from BAL fluid were prepared using aShandon Cytospin 3 (Shandon Scientific LTD, Cheshire, England). Celldifferentials were performed from slides stained with Leukostat (FisherScientific, Pittsburgh, Pa.). Total cell counts were quantified byhemocytometer and, together with the percent type bty differential, wereused to calculate specific cell number.

[0393] Tissue Histology

[0394] Before resection, lungs were inflated with 0.5 ml of 10%phosphate-buffered formalin and fixed overnight at 4° C. The lungsamples were washed free of formalin with 1×PBS and subsequentlydehydrated through an ethanol series prior to equilibration in xyleneand embedded in paraffin. Sections (6μ) were mounted on slides andstained with hematoxylin/eosin, massons trichome and periodicacid-schiff (PAS) reagent. Parasagittal sections were analyzed bybright-field microscopy. Mucus cell content was assessed as the airwayepithelium staining with PAS. Relative comparisons of mucus content weremade between cohorts of animals by counting the number of PAS-positiveairways.

[0395] As shown in FIGS. 11A-11B, B7.2 mRNA (FIG. 11A) and B7.1 mRNA(FIG. 11B) were detected in mouse lung and lymph node during thedevelopment of ovalbumin-induced asthma. Treatment with ISIS 121874following allergen challenge reduces the airway response to methacholine(FIG. 12). The Penh value in B7.2 ASO-treated mice was about 40% lowerthan vehicle-treated mice, and was statistically the same as naive micewhich were not sensitized with the allergen or treated with the ASO.This shows that B7.2 ASO-treated mice had significantly better airflow,and less inflammation, than mice which were not treated with the ASO.The dose-dependent inhibition of the Penh response to methacholine byISIS 121874 is shown in FIG. 13. The inhibition of allergen-inducedeosinophilia by ISIS 121874 is shown in FIG. 14. ISIS 121874 at 0.3mg/kg reduced the total number of eosinophils by about 75% compared tovehicle-treated mice. Since increased numbers of eosinophils result frominflammation, this provides further support for the anti-inflammatoryproperties of the B7.2 ASO. In addition, daily intratracheal delivery ofISIS 121874 does not induce splenomegaly, the concentration of ISIS121874 achieved in lung tissue via daily intratracheal administration isproportional to the dose delivered (FIG. 15) and ISIS 121874 is retainedin lung tissue for at least one week following single dose (0.3 mg/kg)intratracheal administration as determined by capillary gelelectrophoresis (CGE) analysis (FIG. 16).

Example 23

[0396] Support for an Antisense Mechanism of Action for ISIS 121874

[0397] Two variants of ISIS 121874 were synthesized: a 7 base mismatch5′-TCAAGTCCTTCCACACCCAA-3′ (ISIS 306058; SEQ ID NO: 294) and a gapablated oligonucleotide ISIS 306058 having the same sequence as ISIS121'874, but with 2′-MOE modifications at nucleotides 1, 2, 3, 6, 9, 13,16, 18, 19 and 20. Because of the presence of 2′-MOE in the gap, thisoligonucleotide is no longer an RNase H substrate and will not recruitRNase H to the RNA-DNA hybrid which is formed.

[0398] The results (FIG. 17) show that at 0.3 mg/kg, only ISIS 121874,and not the mismatch and gap ablated controls, significantly loweredPenh, which supports that ISIS 121874 is working by an antisensemechanism.

[0399] The effects of ISIS 121874 and the control oligonucleotides onairway mucus production in the ovalbumin-induced model were also tested.The results (FIG. 18) show that only ISIS 121874 significantly inhibitedmucus production.

[0400] The effect of ISIS 121874 on B7.2 and B7.1 mRNA in lung tissue ofallergen-challenged mice is shown in FIGS. 19A and 19B, respectively.The effect of ISIS 121874 on B7.2 and B7.1 mRNA in draining lymph nodesof allergen-challenged mice is shown in FIGS. 20A and 20B, respectively.This shows that ISIS 121874 reduces both B7.2 and B7.1 mRNA (greater inlung vs. node).

[0401] In summary, ISIS 121874 resulted in a dose-dependent inhibitionof airway hypersensitivity, inhibited eosinophilia and reduced B7.1 andB7.2 expression in the lung and lymph nodes. In addition, ISIS 121874reduced levels of the following inflammatory molecules: IQE mRNA in thelung and IgE protein in the serum; reduced IL-5 mRNA in the lung andIL-5 protein in the BAL fluid; and reduced the serum level of macrophagechemokine (KC).

[0402] In the aerosolized ISIS 121874 study, treatment with 0.001, 0.01,0.1 or 1.0 mg/kg estimated inhaled dose was delivered by nose-onlyinhalation of an aerosol solution, four times per day, on days 15-26(n=8 mice per group). The airway response to methacholine was reduced tothe level seen in naive mice at 0.001 mg/kg dose (estimated inhaleddose=1 μg/kg). No gross adverse effects were seen.

Example 24 B7.1 ASO in Ovalbumin Model of Asthma

[0403] The same protocols described above for the B7.2 ASOs were used totest the effect of the B7.1 ASO ISIS 121844 (SEQ ID NO: 286). Incontrast to the B7.2 ASO, ISIS 121844 had no effect on the Penh responsein mice challenged with methacholine. Although there was no effect onPenh, ISIS 121844 reduced allergen-induced airway eosinophilia (FIG. 21)and reduced the levels of B7.1 and B7.2 in the mouse lung. (FIGS.22A-B). Thus, treatment with B7.1 ASO produced anti-inflammatoryeffects, but did not prevent airway hyper-responsiveness. There was noeffect of ISIS 121844 on the Penh response despite achieving an 80%reduction of B7.2 mRNA in the lung (FIG. 21B). Treatment with ISIS121844 reduced eosinophil and PMN numbers in BAL fluid. This effect wasassociated with a reduction in lung B7.2 (not B7.1) mRNA.

[0404] The combined use of B7.1 or B7.2 with one or more conventionalasthma medications including, but not limited to, montelukast sodium(Singulair™), albuterol, beclomethasone dipropionate, triamcinoloneacetonide, ipratropium bromide (Atrovent™); flunisolide, fluticasonepropionate (Flovent™) and other steroids is also contemplated. Thecombined use of oligonucleotides which target both B7.1 and B7.2 for thetreatment of asthma is also within the scope of the present invention.B7.1 and B7.2 may also be combined with one or more conventional asthmamedications as described above for B7.1 or B7.2 alone.

Example 25

[0405] Design and Screening of Duplexed Antisense Compounds TargetingB7.1 or B7.2

[0406] In accordance with the present invention, a series of nucleicacid duplexes comprising the antisense compounds of the presentinvention and their complements can be designed to target B7.1 or B7.2.The nucleobase sequence of the antisense strand of the duplex comprisesat least a portion of an oligonucleotide to B7.1 or B7.2 as describedherein. The ends of the strands may be modified by the addition of oneor more natural or modified nucleobases to form an overhang. The sensestrand of the dsRNA is then designed and synthesized as the complementof the antisense strand and may also contain modifications or additionsto either terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini. For example, a duplexcomprising an antisense strand having the sequence CGAGAGGCGGACGGGACCGand having a two-nucleobase overhang of deoxythymidine (dT) would havethe following structure:   cgagaggcggacgggaccgTT Antisense Strand  ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0407] RNA strands of the duplex can be synthesized by methods disclosedherein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).Once synthesized, the complementary strands are annealed. The singlestrands are aliquoted and diluted to a concentration of 50 uM. Oncediluted, 30 uL of each strand is combined with 15 uL of a 5×solution ofannealing buffer. The final concentration of said buffer is 100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate.The final volume is 75 uL. This solution is incubated for 1 minute at90° C. and then centrifuged for 15 seconds. The tube is allowed to sitfor 1 hour at 37° C. at which time the dsRNA duplexes are used inexperimentation. The final concentration of the dsRNA duplex is 20 uM.This solution can be stored frozen (−20° C.) and freeze-thawed up to 5times.

[0408] Once prepared, the duplexed antisense compounds are evaluated fortheir ability to modulate B7.1 or B7.2 expression according to theprotocols described herein.

Example 26

[0409] Design of Phenotypic Assays and In Vivo Studies for the Use ofB7.1 or B7.2 Inhibitors

[0410] Phenotypic Assays

[0411] Once B7.1 or B7.2 inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

[0412] Phenotypic assays, kits and reagents for their use are well knownto those skilled in the art and are herein used to investigate the roleand/or association of B7.1 or B7.2 in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.) cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays.(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

[0413] In one non-limiting example, cells determined to be appropriatefor a particular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with B7.1 orB7.2 inhibitors identified from the in vitro studies as well as controlcompounds at optimal concentrations which are determined by the methodsdescribed above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

[0414] Phenotypic endpoints include changes in cell morphology over timeor treatment dose as well as changes in levels of cellular componentssuch as proteins, lipids, nucleic acids, hormones, saccharides ormetals. Measurements of cellular status which include pH, stage of thecell cycle, intake or excretion of biological indicators by the cell,are also endpoints of interest.

[0415] Analysis of the genotype of the cell (measurement of theexpression of one or more of the genes of the cell) after treatment isalso used as an indicator of the efficacy or potency of the B7.1 or B7.2inhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

Example 27

[0416] Antisense Inhibition of Human B7.2 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0417] In accordance with the present invention, an additional series ofantisense compounds were designed to target different regions of thehuman B7.2 RNA, using published sequences (GenBark accession numberU04343.1, incorporated herein as SEQ ID NO: 295, GenBank accessionnumber BC040261.1, incorporated herein as SEQ ID NO: 296 and GenBankaccession number NT_(—)005543.12, a portion of which is incorporatedherein as SEQ ID NO: 297). The compounds are shown in Table 25. “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget sequence to which the compound binds. All compounds in Table 25are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human B7.2 mRNA levels in THP-1 cells by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments. If present, “N.D.” indicates “no data”. TABLE 25Inhibition of human B7.2 mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap SEQ Genbank IsisSequence ID % Accession Target Number 5′ to 3′ NO: Targt Site Region322216 ACCAAAAGGAGTATTTGCG 298 N.D. U04343.1 26 5′UTR 322217CATTCCCAAGGAACACAGA 299 N.D. U04343.1 64 5′UTR 322218ACTGTAGCTCCAAAAAGAG 300 N.D. U04343.1 105 5′UTR 322219CTGTCACAAATGCCTGTCC 301 N.D. U04343.1 124 5′UTR 322220TCAGTCCCATAGTGCTGTC 302 N.D. U04343.1 138 START 322221CTGTTACAGCAGCAGAGAA 303 N.D. BC040261.1 29 5′UTR 322222TCCCTGTTACAGCAGCAGA 304 N.D. BC040261.1 32 5′UTR 322223ATCTGCAAATGACCCCACT 305 N.D. BC040261.1 71 5′UTR 322224GTGACCTAATATCTGGAAA 306 N.D. BC040261.1 81 5′UTR 322225CATTTTTGCTGCTTCTGCT 307 N.D. BC040261.1 100 START 322226GGAACTTACAAAGGAAAGG 308 N.D. BC040261.1 1145 3′UTR 322227AAAAAGGTTGCCCAGGAAC 309 N.D. BC040261.1 1159 3′UTR 322228TGCCTTCTGGAAGAAATCA 310 N.D. BC040261.1 1177 3′UTR 322229TTTTTGCCTTCTGGAAGAA 311 N.D. BC040261.1 1181 3′UTR 322230CTATTCCACTTAGAGGGAG 312 N.D. BC040261.1 1233 3′UTR 322231TCTGATCTGGAGGAGGTAT 313 N.D. BC040261.1 1389 3′UTR 322232AGAAATTGAGAGGTCTATT 314 N.D. BC040261.1 1444 3′UTR 322233CACCAGCTTAGAATTCTGG 315 N.D. BC040261.1 1484 3′UTR 322234AGGTAGTTCTTTAGTCACA 316 N.D. BC040261.1 1524 3′UTR 322235CCAGACTGAGGAGGTAGTT 317 N.D. BC040261.1 1535 3′UTR 322236CAGTACATAGATCTCTATG 318 N.D. BC040261.1 1599 3′UTR 322237TTACAGTACATACATCTCT 319 M.D. BC040261.1 1602 3′UTR 322238GATGAGAACTCCTTAGCAG 320 N.D. BC040261.1 1657 3′UTR 322239TAGCAACAGCCCAGATAGA 321 N.D. BC040261.1 1787 3′UTR 322240TCTGTTGCTTGTTTCAAGA 322 N.D. BC040261.1 2043 3′UTR 322241TCCATTTGGACAGACTATC 323 N.D. BC040261.1 2064 3′UTR 322242GGGAAACTGCTGTCTGTCT 324 N.D. BC040261.1 2087 3′UTR 322243TGCTTCCAGGAAGATGACA 325 N.D. BC040261.1 2149 3′UTR 322244ATTCATCCCATTATCAAGG 326 N.D. BC040261.1 2191 3′UTR 322245ACCCAGGAGTCGAAAGTCC 327 N.D. BC040261.1 2223 3′UTR 322246CTTCCTAATTCCGTTGCAG 328 N.D. BC040261.1 2255 3′UTR 322247CATCTGTAGGCTAAGTAAG 329 N.D. BC040261.1 2297 3′UTR 322248CCCGTAGGACATCTGTAGG 330 N.D. BC040261.1 2306 3′UTR 322249GCCCTATCCTGGGCCAGCC 331 N.D. BC040261.1 2331 3′UTR 322250GTCTCTGTATGCAAGTTTC 332 N.D. BC040261.1 2396 3′UTR 322251CCAGTATATCTGTCTCTGT 333 N.D. BC040261.1 2407 3′UTR 322252CCAGGTTTTCAAAGTCATT 334 N.D. BC040261.1 2430 3′UTR 322253AGCCAGGTTTTCAAAGTCA 335 N.D. BC040261.1 2432 3′UTR 322254CCCTTAGTGATCCCACCTT 336 N.D. BC040261.1 2453 3′UTR 322255CTGCCCCATCCCTTAGTGA 337 N.D. BC040261.1 2462 3′UTR 322256TTTATGTTTGGGCAGAGAC 338 N.D. BC040261.1 2480 3′UTR 322257CATGCCAGTCTATAACCCT 339 N.D. BC040261.1 2556 3′UTR 322258TAGCATGGCAGTCTATAAC 340 N.D. BC040261.1 2559 3′UTR 322259TCTAGCATGGCAGTCTATA 341 N.D. BC040261.1 2561 3′UTR 322260TTGTCTAGCATGGCAGTCT 342 N.D. BC040261.1 2564 3′UTR 322261AAGCTTGTCTAGCATGGCA 343 N.D. BC040261.1 2568 3′UTR 322262ACATGGACAAGCTTGTCTA 344 N.D. BC040261.1 2576 3′UTR 322263TTACATGGACAAGCTTGTC 345 N.D. BC040261.1 2578 3′UTR 322264GAATATTACATGGACAAGC 346 N.D. BC040261.1 2583 3′UTR 322265AACTAGCCAGGTGCTAGGA 347 N.D. BC040261.1 2636 3′UTR 322266AATTATTACTCACCACTGC 348 N.D. NT_005543.12 1124 genomic 322267TAATATTTAGGCAAGCATG 349 N.D. NT_005543.12 13890 genomic 322268GGACCCTGGGCCAGTTATT 350 N.D. NT_005543.12 22504 genomic 322269CAAACATACCTGTCACAAA 351 N.D. NT_005543.12 23662 genomic 322270CTCATATCAATTGATGGCA 352 N.D. NT_005543.12 29265 genomic 322271TGCTACATCTACTCAGTGT 353 N.D. NT_005543.12 31796 genomic 322272TGGAAACTCTTGCCTTCGG 354 N.D. NT_005543.12 32971 genomic 322273CCATCCACATTGTAGCATG 355 N.D. NT_005543.12 34646 genomic 322274TCAGGATGGTATGGCCATA 356 N.D. NT_005543.12 36251 genomic 322275TCCCATAGTGCTAGAGTCG 357 N.D. NT_005543.12 37218 genomic 322276AGGTTCTTACCAGAGAGCA 358 N.D. NT_005543.12. 37268 genomic 322277CAGAGGAGCACCACCTAAA 359 N.D. NT_005543.12 49133 genomic 322278GACCACATACCAAGCACTG 360 N.D. NT_005543.12 49465 genomic 322279ATCTTTCAGAAACCCAAGC 361 N.D. NT_005543.12 51347 genomic 322280GAGTCACCAAAGATTTACA 362 N.D. NT_005543.12 51542 genomic 322281CTGAAGTTAGCTGAAAGCA 363 N.D. NT_005543.12 51815 genomic 322282ACAGCTTTACCTATAGACA 364 N.D. NT_005543.12 52118 genomic 322283TCCTCAAGCTCTACAAATG 365 N.D. NT_005543.12 54882 genornic 322284GACTCACTCACCACATTTA 366 N.D. NT_005543.12 55027 genomic 322285AGTGATACCAAGGCTTCTC 367 N.D. NT_005543.12 56816 genomic 322286CTTGGAGAGAATGGTTATC 368 N.D. NT_005543.12 61044 genomic 322287GAAGATGTTGATGCCTAAA 369 N.D. NT_005543.12 63271 genomic 322288GTGTTGGTTCCTGAAAGAC 370 N.D. NT_005543.12 63665 genomic 322289CAGGATTTACCTTTTCTTC 371 N.D. NT_005543.12 63711 genomic 322290AGGGCAGAATAGAGGTTGC 372 N.D. NT_005543.12 64973 Genomic 322291TTTTTCTCTGGAGAAATAG 373 N.D. NT_005543.12 65052 genomic 323624GTTACTCAGTCCCATAGTG 374 59 U04343.1 143 START 323625 CAAAGAGAATGTTACTCAG375 21 U04343.1 153 Coding 323626 CCATCACAAAGAGAATGTT 376 32 U04343.1159 Coding 323627 GGAAGGCCATCACAAAGAG 377 54 U04343.1 165 Coding 323628GAGCAGGAAGGCCATCACA 378 44 U04343.1 170 Coding 323629CCAGAGAGCAGGAAGGCCA 379 36 U04343.1 175 Coding 323630AAATAAGCTTGAATCTTCA 380 22 U04343.1 205 Coding 323631AGTCTCATTGAAATAAGCT 381 56 U04343.1 215 Coding 323632AGGTCTGCAGTCTCATTGA 382 41 U04343.1 223 Coding 323633CTACTAGCTCACTCAGGCT 383 50 U04343.1 273 Coding 323634AAATACTACTAGCTCACTC 384 30 U04343.1 278 Coding 323635CTGCCAAAATACTACTAGC 385 24 U04343.1 284 Coding 323636TTCAGAACCAAGTTTTCCT 386 23 U04343.1 307 Coding 323637CCTCATTCAGAACCAAGTT 387 19 U04343.1 312 Coding 323638GTATACCTCATTCAGAACC 388 20 U04343.1 317 Coding 323639GCCTAAGTATACCTCATTC 389 55 U04343.1 323 Coding 323640CTCTTTGCCTAAGTATACC 390 28 U04343.1 329 Coding 323641CCCATATACTTGGAATGAA 391 88 U04343.1 361 Coding 323642CTTGTGCGGCCCATATACT 392 27 U04343.1 370 Coding 323643ATCAAAACTTGTGCGGCCC 393 80 U04343.1 377 Coding 323644CCCTTGTCCTTGATCTGAA 394 71 U0.4343.1 427 Coding 323645ACAAGCCCTTGTCCTTGAT 395 56 U04343.1 432 Coding 323646TTGATACAAGCCCTTGTCC 396 33 U04343.1 437 Coding 323647ATACATTGATACAAGCCCT 397 41 U04343.1 442 Coding 323648TGGATGATACATTGATACA 398 31 U04343.1 448 Coding 323649GAATTCATCTGGTGGATGC 399 81 U04343.1 493 Coding 323650GTTCACAATTCATCTGGTG 400 92 U04343.1 498 Coding 323651TGACAGTTCAGAATTCATC 401 64 U04343.1 503 Coding 323652AGCACTGACAGTTCAGAAT 402 87 U04343.1 508 Coding 323653TAGCAAGCACTGACAGTTC 403 96 U04343.1 513 Coding 323654TGAAGTTAGCAAGCACTGA 404 87 U04343.1 519 Coding 323655TTGACTGAAGTTAGCAAGC 405 65 U04343.1 524 Coding 323656CTATTTCAGGTTGACTGAA 406 76 U04343.1 534 Coding 323657TCTGTTATATTAGAAATTG 407 43 U04343.1 556 Coding 323658GCAGGTCAAATTTATGTAC 408 36 U04343.1 581 Coding 323659GTATAGATGAGCAGGTCAA 409 56 U04343.1 591 Coding 323660GGGTAACCGTGTATAGATG 410 71 U04343.1 601 Coding 323661AGGTTCTGGGTAACCGTGT 411 68 U04343.1 608 Coding 323662TAGCAAAACACTCATCTTC 412 22 U04343.1 629 Coding 323663 GTTCTTAGCAAAACACTCA413 23 U04343.1 634 Coding 323664 ATTCTTGGTTCTTAGCAAA 414 35 U04343.1641 Coding 323665 GATAdTTGAATTCTTGGTT 415 43 U04343.1 650 Coding 323666ACCATCATACTCGATAGTT 416 71 U04343.1 662 Coding 323667ATCTTGAGATTTCTGCATA 417 52 U04343.1 683 Coding 323668ACATTATCTTCAGATTTCT 418 39 U04343.1 688 Coding 323669 CGTACACTTCTGTGACATT419 68 U04343.1 702 Coding 323670 AGACAAGCTGATGGAAACG 420 19 U04343.1722 Coding 323671 GAAACAGACAAGCTGATGG 421 26 U04343.1 727 Coding 323672GGAATGAAACAGACAAGCT 422 33 U043431 732 Coding 323673 CATCAGGGAATGAAACAGA423 38 U04343.1 738 Coding 323674 CGTAACATCAGGGAATGAA 424 47 U04343.1743 Coding 323675 AGCTCTATAGAGAAAGGTG 425 77 U04343.1 817 Coding 323676CCTCAAGCTCTATAGAGAA 426 24 U04343.1 822 Coding 323677GGAGGCTGAGGGTCCTCAA 427 55 U04343.1 835 Coding 323678AGTACAGCTGTAATCCAAG 428 23 U04343.1 868 Coding 323679TTGGAAGTACAGCTGTAAT 429 60 U04343.1 873 Coding 323680ATAATAACTGTTGGAAGTA 430 51 U04343.1 883 Coding 323681CATCACACATATAATAACT 431 8 U04343.1 893 Coding 323682 TCCATTTCCATAGAATTAG432 35 U04343.1 921 Coding 323683 TCTTCTTCCATTTCCATAG 433 16 U04343.1927 Coding 323684 ATTTATAACAGTTGCGAGG 434 32 U04343.1 954 Coding 323685TTGGTTCCACATTTATAAG 435 18 U04343.1 964 Coding 323686CTCTCCATTGTGTTGGTTC 436 53 U04343.1 976 Coding 323687CTTCCCTCTCCATTGTGTT 437 19 U04343.1 981 Coding 323688TGGTCTGTTCACTCTCTTC 438 58 U04343.1 996 Coding 323689TTCATCAGATCTTTCAGGT 439 43 U04343.1 1037 Coding 323690ATCACTTTTGTCGCATGAA 440 82 U04343.1 1088 Coding 323691GCTTTACTCTTTAATTAAA 441 40 U04343.1 1114 STOP 323692 GTATGOGCTTTACTCTTTA442 5 7 U04343.1 1120 3′UTR 323693 ATACTTGTATGOCCTTTAC 443 62 U04343.11126 3′UTR 323694 AATGAATACTTGTATGGGC 444 71 U04343.1 1131 3′UTR

[0418]

1 444 1 32 DNA Artificial Sequence Synthetic 1 gatcagggta ccaggagccttaggaggtac gg 32 2 33 DNA Artificial Sequence Synthetic 2 gatagcctcgagttatttcc aggtcatgag cca 33 3 20 DNA Artificial Sequence Synthetic 3ttccaggtca tgagccatta 20 4 21 DNA Artificial Sequence Synthetic 4cataaggtgt gctctgaagt g 21 5 20 DNA Artificial Sequence Synthetic 5ttactcatgg taatgtcttt 20 6 20 DNA Artificial Sequence Synthetic 6attaaaaaca tgtatcactt 20 7 21 DNA Artificial Sequence Synthetic 7ggaacacaga agcaaggtgg t 21 8 20 DNA Artificial Sequence Synthetic 8ccgtacctcc taaggctcct 20 9 20 DNA Artificial Sequence Synthetic 9cccatagtgc tgtcacaaat 20 10 20 DNA Artificial Sequence Synthetic 10gcacagcagc attcccaagg 20 11 20 DNA Artificial Sequence Synthetic 11ttgcaaattg gcatggcagg 20 12 20 DNA Artificial Sequence Synthetic 12tggtatgggc tttactcttt 20 13 20 DNA Artificial Sequence Synthetic 13aaaaggttgc ccaggaacgg 20 14 20 DNA Artificial Sequence Synthetic 14gggagtcctg gagccccctt 20 15 20 DNA Artificial Sequence Synthetic 15ccattaagct gggcttggcc 20 16 20 DNA Artificial Sequence Synthetic 16tgcgagctcc ccgtacctcc 20 17 20 DNA Artificial Sequence Synthetic 17gcccaagctg gcatccgtca 20 18 20 DNA Artificial Sequence Synthetic 18ggattgccaa gcccatggtg 20 19 20 DNA Artificial Sequence Synthetic 19ctaagtagtg ctagccggga 20 20 38 DNA Artificial Sequence Synthetic 20gatcagggta ccccaaagaa aaagtgattt gtcattgc 38 21 35 DNA ArtificialSequence Synthetic 21 gatagcctcg aggataatga attggctgac aagac 35 22 20DNA Artificial Sequence Synthetic 22 gggtaagact ccacttctga 20 23 20 DNAArtificial Sequence Synthetic 23 gggtctccaa aggttgtgga 20 24 20 DNAArtificial Sequence Synthetic 24 gttcctgggt ctccaaaggt 20 25 20 DNAArtificial Sequence Synthetic 25 acacacagag attggagggt 20 26 20 DNAArtificial Sequence Synthetic 26 gctcacgtag aagaccctcc 20 27 20 DNAArtificial Sequence Synthetic 27 ggcagggctg atgacaatcc 20 28 20 DNAArtificial Sequence Synthetic 28 tgcaaaacag gcagggctga 20 29 20 DNAArtificial Sequence Synthetic 29 agaccagggc acttcccagg 20 30 20 DNAArtificial Sequence Synthetic 30 cctgcctccg tgtgtggccc 20 31 20 DNAArtificial Sequence Synthetic 31 gaccagccag caccaagagc 20 32 20 DNAArtificial Sequence Synthetic 32 ccacaggaca gcgttgccac 20 33 20 DNAArtificial Sequence Synthetic 33 ccggttcttg tactcgggcc 20 34 20 DNAArtificial Sequence Synthetic 34 ccaaccagga gaggtgaggc 20 35 20 DNAArtificial Sequence Synthetic 35 ggcaaagcag taggtcaggc 20 36 20 DNAArtificial Sequence Synthetic 36 gcctcatgat ccccacgatc 20 37 20 DNAArtificial Sequence Synthetic 37 agtcctacta ccagccgcct 20 38 20 DNAArtificial Sequence Synthetic 38 tcagggtaag actccacttc 20 39 20 DNAArtificial Sequence Synthetic 39 agggtgttcc tgggtctcca 20 40 20 DNAArtificial Sequence Synthetic 40 ctccgtgtgt ggcccatggc 20 41 20 DNAArtificial Sequence Synthetic 41 ggatggtgat gttccctgcc 20 42 20 DNAArtificial Sequence Synthetic 42 tgagaaagac cagccagcac 20 43 20 DNAArtificial Sequence Synthetic 43 gggcgcagag ccaggatcac 20 44 20 DNAArtificial Sequence Synthetic 44 ggcccaggat gggagcaggt 20 45 20 DNAArtificial Sequence Synthetic 45 agggcgtaca ctttcccttc 20 46 20 DNAArtificial Sequence Synthetic 46 cagccccttg cttctgcgga 20 47 20 DNAArtificial Sequence Synthetic 47 aaggagaggg atgccagcca 20 48 22 DNAArtificial Sequence Synthetic 48 ctgttacttt acagagggtt tg 22 49 25 DNAArtificial Sequence Synthetic 49 cttctgttac tttacagagg gtttg 25 50 21DNA Artificial Sequence Synthetic 50 ctgttacttt acagagggtt t 21 51 20DNA Artificial Sequence Synthetic 51 gccctcgtca gatgggcgca 20 52 20 DNAArtificial Sequence Synthetic 52 agtcctacta ccagccgcct 20 53 20 DNAArtificial Sequence Synthetic 53 agtaagagtc tattgaggta 20 54 20 DNAArtificial Sequence Synthetic 54 ggttgagttt cacaacctga 20 55 20 DNAArtificial Sequence Synthetic 55 gtccacagaa tggaacagag 20 56 20 DNAArtificial Sequence Synthetic 56 ggcatccacc cggcagatgc 20 57 20 DNAArtificial Sequence Synthetic 57 tggatggcat ccacccggca 20 58 20 DNAArtificial Sequence Synthetic 58 aggcacctcc taggctcaca 20 59 20 DNAArtificial Sequence Synthetic 59 gccaatggag cttaggcacc 20 60 20 DNAArtificial Sequence Synthetic 60 catgatgggg aaagccagga 20 61 20 DNAArtificial Sequence Synthetic 61 aattgcaagc catagcttca 20 62 20 DNAArtificial Sequence Synthetic 62 cggcaaggca gcaatacctt 20 63 20 DNAArtificial Sequence Synthetic 63 cccagcaatg acagacagca 20 64 20 DNAArtificial Sequence Synthetic 64 ggtctgaaag gaccaggccc 20 65 20 DNAArtificial Sequence Synthetic 65 tgggaaaccc ccggaagcaa 20 66 20 DNAArtificial Sequence Synthetic 66 ggctttggga aacccccgga 20 67 19 DNAArtificial Sequence Synthetic 67 tcagattcag gatctggga 19 68 20 DNAArtificial Sequence Synthetic 68 cccaggtgaa gtcctctgac 20 69 20 DNAArtificial Sequence Synthetic 69 ctgcgccgaa tcctgcccca 20 70 20 DNAArtificial Sequence Synthetic 70 caggcccgaa ggtaaggctg 20 71 20 DNAArtificial Sequence Synthetic 71 tcagctagca cggtgctgaa 20 72 20 DNAArtificial Sequence Synthetic 72 ggcccagcaa acttgcccgt 20 73 20 DNAArtificial Sequence Synthetic 73 ccaccacagt gggctcagcc 20 74 19 DNAArtificial Sequence Synthetic 74 ggccatgagg gcaatctaa 19 75 21 DNAArtificial Sequence Synthetic 75 gtggccatga gggcaatcta a 21 76 20 DNAArtificial Sequence Synthetic 76 gatttaacat ttggcgccca 20 77 20 DNAArtificial Sequence Synthetic 77 aaagttacaa cattatatct 20 78 20 DNAArtificial Sequence Synthetic 78 agtgcgattc tcaaacctac 20 79 16 DNAArtificial Sequence Synthetic 79 tatttgcgag ctcccc 16 80 15 DNAArtificial Sequence Synthetic 80 tatttgcgag ctccc 15 81 14 DNAArtificial Sequence Synthetic 81 tatttgcgag ctcc 14 82 20 DNA ArtificialSequence Synthetic 82 cgacagctcc tgcgctcctc 20 83 16 DNA ArtificialSequence Synthetic 83 agctccccgt acctcc 16 84 16 DNA Artificial SequenceSynthetic 84 tgcgagctcc ccgtac 16 85 10 DNA Artificial SequenceSynthetic 85 ctccccgtac 10 86 11 DNA Artificial Sequence Synthetic 86gctccccgta c 11 87 12 DNA Artificial Sequence Synthetic 87 agctccccgt ac12 88 13 DNA Artificial Sequence Synthetic 88 gagctccccg tac 13 89 14DNA Artificial Sequence Synthetic 89 cgagctcccc gtac 14 90 15 DNAArtificial Sequence Synthetic 90 gcgagctccc cgtac 15 91 13 DNAArtificial Sequence Synthetic 91 gcgagctccc cgt 13 92 15 DNA ArtificialSequence Synthetic 92 gccgccgcca agtct 15 93 24 DNA Artificial SequenceSynthetic 93 gagaagcaaa gctttcaccc tgtg 24 94 22 DNA Artificial SequenceSynthetic 94 gaagcaaagc tttcaccctg tg 22 95 19 DNA Artificial SequenceSynthetic 95 gcaaagcttt caccctgtg 19 96 24 DNA Artificial SequenceSynthetic 96 ctccccgtac ctcctaaggc tcct 24 97 22 DNA Artificial SequenceSynthetic 97 ccccgtacct cctaaggctc ct 22 98 19 DNA Artificial SequenceSynthetic 98 ccgtacctcc taaggctcc 19 99 32 DNA Artificial SequenceSynthetic 99 gatcagggta ccaagagtgg ctcctgtagg ca 32 100 32 DNAArtificial Sequence Synthetic 100 gatagcctcg aggtagaatt ccaatcagct ga 32101 20 DNA Artificial Sequence Synthetic 101 tgcatccccc aggccaccat 20102 21 DNA Artificial Sequence Synthetic 102 gccgaggtcc atgtcgtacg c 21103 20 DNA Artificial Sequence Synthetic 103 acacgtctac aggagtctgg 20104 20 DNA Artificial Sequence Synthetic 104 caagcccatg gtgcatctgg 20105 20 DNA Artificial Sequence Synthetic 105 ctggggtcca tcgtgggtgc 20106 20 DNA Artificial Sequence Synthetic 106 ccgtgctgcc tacaggagcc 20107 20 DNA Artificial Sequence Synthetic 107 ggtgcttccg taagttctgg 20108 20 DNA Artificial Sequence Synthetic 108 ggattgccaa gcccatggtg 20109 20 DNA Artificial Sequence Synthetic 109 ctaagtagtg ctagccggga 20110 20 DNA Artificial Sequence Synthetic 110 tgcgtctcca cggaaacagc 20111 20 DNA Artificial Sequence Synthetic 111 gtgcggccca ggtacttggc 20112 20 DNA Artificial Sequence Synthetic 112 acaaggagga gggccacagt 20113 20 DNA Artificial Sequence Synthetic 113 tgagaggttt ggaggaaatc 20114 20 DNA Artificial Sequence Synthetic 114 gatagtctct ctgtcagcgt 20115 20 DNA Artificial Sequence Synthetic 115 gttgctgggc ctgctaggct 20116 20 DNA Artificial Sequence Synthetic 116 ctaggtctcg tcgtcggtgg 20117 20 DNA Artificial Sequence Synthetic 117 tctcactgcc ttcactctgc 20118 21 DNA Artificial Sequence Synthetic 118 gtaccagatg aaggttatca a 21119 20 DNA Artificial Sequence Synthetic 119 ctttggagat tattcgagtt 20120 20 DNA Artificial Sequence Synthetic 120 gcaagtgtaa agccctgagt 20121 20 DNA Artificial Sequence Synthetic 121 agaattccaa tcagctgaga 20122 20 DNA Artificial Sequence Synthetic 122 tctgagaaac tctgcacttc 20123 20 DNA Artificial Sequence Synthetic 123 tcctcaggct ctcactgcct 20124 20 DNA Artificial Sequence Synthetic 124 ggttgttcaa gtccgtgctg 20125 21 DNA Artificial Sequence Synthetic 125 gccgaggtcc atgtcgtagc c 21126 20 DNA Artificial Sequence Synthetic 126 agactccact tctgagatgt 20127 20 DNA Artificial Sequence Synthetic 127 tgaagaaaaa ttccactttt 20128 20 DNA Artificial Sequence Synthetic 128 tttagtttca cagcttgctg 20129 20 DNA Artificial Sequence Synthetic 129 tcccaggtgc aaaacaggca 20130 20 DNA Artificial Sequence Synthetic 130 gtgaaagcca acaatttgga 20131 20 DNA Artificial Sequence Synthetic 131 catggcttca gatgcttagg 20132 20 DNA Artificial Sequence Synthetic 132 ttgaggtatg gacacttgga 20133 20 DNA Artificial Sequence Synthetic 133 gcgttgccac ttctttcact 20134 20 DNA Artificial Sequence Synthetic 134 ttttgccagt agatgcgagt 20135 20 DNA Artificial Sequence Synthetic 135 ggccatatat tcatgtcccc 20136 20 DNA Artificial Sequence Synthetic 136 gccaggatca caatggagag 20137 20 DNA Artificial Sequence Synthetic 137 gtatgtgccc tcgtcagatg 20138 20 DNA Artificial Sequence Synthetic 138 ttcagccagg tgttcccgct 20139 20 DNA Artificial Sequence Synthetic 139 ggaagtcagc tttgactgat 20140 20 DNA Artificial Sequence Synthetic 140 cctccagagg ttgagcaaat 20141 20 DNA Artificial Sequence Synthetic 141 ccaaccagga gaggtgaggc 20142 20 DNA Artificial Sequence Synthetic 142 gaagctgtgg ttggttgtca 20143 20 DNA Artificial Sequence Synthetic 143 ttgaaggtct gattcactct 20144 20 DNA Artificial Sequence Synthetic 144 aaggtaatgg cccaggatgg 20145 20 DNA Artificial Sequence Synthetic 145 aagcagtagg tcaggcagca 20146 20 DNA Artificial Sequence Synthetic 146 ccttgcttct gcggacactg 20147 20 DNA Artificial Sequence Synthetic 147 agccccttgc ttctgcggac 20148 20 DNA Artificial Sequence Synthetic 148 tgacggaggc taccttcaga 20149 20 DNA Artificial Sequence Synthetic 149 gtaaaacagc ttaaatttgt 20150 20 DNA Artificial Sequence Synthetic 150 agaagaggtt acattaagca 20151 20 DNA Artificial Sequence Synthetic 151 agataatgaa ttggctgaca 20152 20 DNA Artificial Sequence Synthetic 152 gcgtcatcat ccgcaccatc 20153 20 DNA Artificial Sequence Synthetic 153 cgttgcttgt gccgacagtg 20154 20 DNA Artificial Sequence Synthetic 154 gctcacgaag aacaccttcc 20155 20 DNA Artificial Sequence Synthetic 155 agagaaacta gtaagagtct 20156 20 DNA Artificial Sequence Synthetic 156 tggcatccac ccggcagatg 20157 20 DNA Artificial Sequence Synthetic 157 tcgagaaaca gagatgtaga 20158 20 DNA Artificial Sequence Synthetic 158 tggagcttag gcacctccta 20159 20 DNA Artificial Sequence Synthetic 159 tggggaaagc caggaatcta 20160 20 DNA Artificial Sequence Synthetic 160 cagcacaaag agaagaatga 20161 20 DNA Artificial Sequence Synthetic 161 atgaggagag ttgtaacggc 20162 20 DNA Artificial Sequence Synthetic 162 aagtccggtt cttatactcg 20163 20 DNA Artificial Sequence Synthetic 163 gcaggtaatc cttttagtgt 20164 20 DNA Artificial Sequence Synthetic 164 gtgaagtcct ctgacacgtg 20165 20 DNA Artificial Sequence Synthetic 165 cgaatcctgc cccaaagagc 20166 20 DNA Artificial Sequence Synthetic 166 actgcgccga atcctgcccc 20167 20 DNA Artificial Sequence Synthetic 167 ttgatgatga caacgatgac 20168 20 DNA Artificial Sequence Synthetic 168 ctgttgtttg tttctctgct 20169 20 DNA Artificial Sequence Synthetic 169 tgttcagcta atgcttcttc 20170 20 DNA Artificial Sequence Synthetic 170 gttaactcta tcttgtgtca 20171 20 DNA Artificial Sequence Synthetic 171 tccacttcag tcatcaagca 20172 20 DNA Artificial Sequence Synthetic 172 tgctcaatac tctcttttta 20173 20 DNA Artificial Sequence Synthetic 173 aggcccagca aacttgcccg 20174 20 DNA Artificial Sequence Synthetic 174 aacggcaagg cagcaatacc 20175 20 DNA Artificial Sequence Synthetic 175 cagaagcaag gtggtaagaa 20176 20 DNA Artificial Sequence Synthetic 176 gcctgtccac tgtagctcca 20177 20 DNA Artificial Sequence Synthetic 177 agaatgttac tcagtcccat 20178 20 DNA Artificial Sequence Synthetic 178 tcagaggagc agcaccagag 20179 20 DNA Artificial Sequence Synthetic 179 tggcatggca ggtctgcagt 20180 20 DNA Artificial Sequence Synthetic 180 agctcactca ggctttggtt 20181 20 DNA Artificial Sequence Synthetic 181 tgcctaagta tacctcattc 20182 20 DNA Artificial Sequence Synthetic 182 ctgtcaaatt tctctttgcc 20183 20 DNA Artificial Sequence Synthetic 183 catatacttg gaatgaacac 20184 20 DNA Artificial Sequence Synthetic 184 ggtccaactg tccgaatcaa 20185 20 DNA Artificial Sequence Synthetic 185 tgatctgaag attgtgaagt 20186 20 DNA Artificial Sequence Synthetic 186 aagcccttgt ccttgatctg 20187 20 DNA Artificial Sequence Synthetic 187 tgtgatggat gatacattga 20188 20 DNA Artificial Sequence Synthetic 188 tcaggttgac tgaagttagc 20189 20 DNA Artificial Sequence Synthetic 189 gtgtatagat gagcaggtca 20190 20 DNA Artificial Sequence Synthetic 190 tctgtgacat tatcttgaga 20191 20 DNA Artificial Sequence Synthetic 191 aagataaaag ccgcgtcttg 20192 20 DNA Artificial Sequence Synthetic 192 agaaaaccat cacacatata 20193 20 DNA Artificial Sequence Synthetic 193 agagttgcga ggccgcttct 20194 20 DNA Artificial Sequence Synthetic 194 tccctctcca ttgtgttggt 20195 20 DNA Artificial Sequence Synthetic 195 catcagatct ttcaggtata 20196 20 DNA Artificial Sequence Synthetic 196 ggctttactc tttaattaaa 20197 20 DNA Artificial Sequence Synthetic 197 gaaatcaaaa aggttgccca 20198 20 DNA Artificial Sequence Synthetic 198 ggagtcctgg agccccctta 20199 20 DNA Artificial Sequence Synthetic 199 ttggcatacg gagcagagct 20200 20 DNA Artificial Sequence Synthetic 200 tgtgctctga agtgaaaaga 20201 20 DNA Artificial Sequence Synthetic 201 ggcttggccc ataagtgtgc 20202 20 DNA Artificial Sequence Synthetic 202 cctaaatttt atttccaggt 20203 20 DNA Artificial Sequence Synthetic 203 gctccaagtg tcccaatgaa 20204 20 DNA Artificial Sequence Synthetic 204 agtatgtttc tcactccgat 20205 20 DNA Artificial Sequence control oligonucleotide 205 tgccagcacccggtacgtcc 20 206 20 DNA Artificial Sequence Synthetic 206 gctgcctacaggagccactc 20 207 20 DNA Artificial Sequence Synthetic 207 tcaagtccgtgctgcctaca 20 208 20 DNA Artificial Sequence Synthetic 208 gtctacaggagtctggttgt 20 209 20 DNA Artificial Sequence Synthetic 209 agcttgcgtctccacggaaa 20 210 20 DNA Artificial Sequence Synthetic 210 tcacactatcaagtttctct 20 211 20 DNA Artificial Sequence Synthetic 211 gtcaaagctcgtgcggccca 20 212 20 DNA Artificial Sequence Synthetic 212 gtgaagtcgtagagtccagt 20 213 20 DNA Artificial Sequence Synthetic 213 gtgaccttgcttagacgtgc 20 214 20 DNA Artificial Sequence Synthetic 214 catcttcttaggtttcgggt 20 215 20 DNA Artificial Sequence Synthetic 215 ggctgttggagatactgaac 20 216 20 DNA Artificial Sequence Synthetic 216 gggaatgaaagagagaggct 20 217 20 DNA Artificial Sequence Synthetic 217 acatacaatgatgagcagca 20 218 20 DNA Artificial Sequence Synthetic 218 gtctctctgtcagcgttact 20 219 20 DNA Artificial Sequence Synthetic 219 tgccaagcccatggtgcatc 20 220 20 DNA Artificial Sequence Synthetic 220 gcaatttggggttcaagttc 20 221 20 DNA Artificial Sequence Synthetic 221 caatcagctgagaacatttt 20 222 20 DNA Artificial Sequence Synthetic 222 ttttgtataaaacaatcata 20 223 20 DNA Artificial Sequence Synthetic 223 ccttcactctgcatttggtt 20 224 20 DNA Artificial Sequence Synthetic 224 tgcatgttatcaccatactc 20 225 20 DNA Artificial Sequence Synthetic 225 ccctccagtgatgtttacaa 20 226 20 DNA Artificial Sequence Synthetic 226 gaagaccctccagtgatgtt 20 227 20 DNA Artificial Sequence Synthetic 227 cgtagaagaccctccagtga 20 228 20 DNA Artificial Sequence Synthetic 228 ttcccaggtgcaaaacaggc 20 229 20 DNA Artificial Sequence Synthetic 229 tggcttcagatgcttagggt 20 230 20 DNA Artificial Sequence Synthetic 230 cctccgtgtgtggcccatgg 20 231 20 DNA Artificial Sequence Synthetic 231 ggtgatgttccctgcctccg 20 232 20 DNA Artificial Sequence Synthetic 232 gatggtgatgttccctgcct 20 233 20 DNA Artificial Sequence Synthetic 233 aggtatggacacttggatgg 20 234 20 DNA Artificial Sequence Synthetic 234 gaaagaccagccagcaccaa 20 235 20 DNA Artificial Sequence Synthetic 235 cagcgttgccacttctttca 20 236 20 DNA Artificial Sequence Synthetic 236 gtgaccacaggacagcgttg 20 237 20 DNA Artificial Sequence Synthetic 237 agatgcgagtttgtgccagc 20 238 20 DNA Artificial Sequence Synthetic 238 ccttttgccagtagatgcga 20 239 20 DNA Artificial Sequence Synthetic 239 cggttcttgtactcgggcca 20 240 20 DNA Artificial Sequence Synthetic 240 cgcagagccaggatcacaat 20 241 20 DNA Artificial Sequence Synthetic 241 cttcagccaggtgttcccgc 20 242 20 DNA Artificial Sequence Synthetic 242 taacgtcacttcagccaggt 20 243 20 DNA Artificial Sequence Synthetic 243 ttctccattttccaaccagg 20 244 20 DNA Artificial Sequence Synthetic 244 ctgttgtgttgatggcattt 20 245 20 DNA Artificial Sequence Synthetic 245 catgaagctgtggttggttg 20 246 20 DNA Artificial Sequence Synthetic 246 aggaaaatgctcttgcttgg 20 247 20 DNA Artificial Sequence Synthetic 247 tgggagcaggttatcaggaa 20 248 20 DNA Artificial Sequence Synthetic 248 taaggtaatggcccaggatg 20 249 20 DNA Artificial Sequence Synthetic 249 ggtcaggcagcatatcacaa 20 250 20 DNA Artificial Sequence Synthetic 250 gccccttgcttgtgcggaca 20 251 20 DNA Artificial Sequence Synthetic 251 agatcttttcagccccttgc 20 252 20 DNA Artificial Sequence Synthetic 252 tttgttaagggaagaatgcc 20 253 20 DNA Artificial Sequence Synthetic 253 aaaggagagggatgccagcc 20 254 20 DNA Artificial Sequence Synthetic 254 caagacaattcaagatggca 20 255 20 DNA Artificial Sequence Synthetic 255 cgtgtgtctgtgctagtccc 20 256 20 DNA Artificial Sequence Synthetic 256 gctgcttctgctgtgaccta 20 257 20 DNA Artificial Sequence Synthetic 257 tatttgcgagctccccgtac 20 258 20 DNA Artificial Sequence Synthetic 258 gcataagcacagcagcattc 20 259 20 DNA Artificial Sequence Synthetic 259 tccaaaaagagaccagatgc 20 260 20 DNA Artificial Sequence Synthetic 260 aaatgcctgtccactgtagc 20 261 20 DNA Artificial Sequence Synthetic 261 cttcagaggagcagcaccag 20 262 20 DNA Artificial Sequence Synthetic 262 gaatcttcagaggagcagca 20 263 20 DNA Artificial Sequence Synthetic 263 caaattggcatggcaggtct 20 264 20 DNA Artificial Sequence Synthetic 264 gctttggttttgagagtttg 20 265 20 DNA Artificial Sequence Synthetic 265 aggctttggttttgagagtt 20 266 20 DNA Artificial Sequence Synthetic 266 gctcactcaggctttggttt 20 267 20 DNA Artificial Sequence Synthetic 267 ggtcctgccaaaatactact 20 268 20 DNA Artificial Sequence Synthetic 268 agcccttgtccttgatctga 20 269 20 DNA Artificial Sequence Synthetic 269 tgtgggctttttgtgatgga 20 270 20 DNA Artificial Sequence Synthetic 270 aatcattcctgtgggctttt 20 271 20 DNA Artificial Sequence Synthetic 271 ccgtgtatagatgagcaggt 20 272 20 DNA Artificial Sequence Synthetic 272 accgtgtatagatgagcagg 20 273 20 DNA Artificial Sequence Synthetic 273 tcatcttcttaggttctggg 20 274 20 DNA Artificial Sequence Synthetic 274 acaagctgatggaaacgtcg 20 275 20 DNA Artificial Sequence Synthetic 275 tgctcgtaacatcagggaat 20 276 20 DNA Artificial Sequence Synthetic 276 aagatggtcatattgctcgt 20 277 20 DNA Artificial Sequence Synthetic 277 cgcgtcttgtcagtttccag 20 278 20 DNA Artificial Sequence Synthetic 278 cagctgtaatccaaggaatg 20 279 20 DNA Artificial Sequence Synthetic 279 gggcttcatcagatctttca 20 280 20 DNA Artificial Sequence Synthetic 280 catgtatcacttttgtcgca 20 281 20 DNA Artificial Sequence Synthetic 281 agcccccttattactcatgg 20 282 20 DNA Artificial Sequence Synthetic 282 ggagttacagccaggctatt 20 283 20 DNA Artificial Sequence Synthetic 283 agtctcctcttggcatacgg 20 284 20 DNA Artificial Sequence Synthetic 284 cccataagtgtgctctgaag 20 285 20 DNA Artificial Sequence Synthetic 285 tccgtcatcgctcctcaggg 20 286 20 DNA Artificial Sequence Synthetic 286 gctcagcctttccacttcag 20 287 20 DNA Artificial Sequence Synthetic 287gctcagcctttccacttcag 20 288 19 DNA Artificial Sequence Synthetic 288ggccctcctc cttgtgatg 19 289 29 DNA Artificial Sequence Synthetic 289tgctcatcat tgtatgtcac aagaagccg 29 290 19 DNA Artificial SequenceSynthetic 290 ctgggcctgc taggctgat 19 291 20 DNA Artificial SequenceSynthetic 291 caggaagcta cgggcaagtt 20 292 27 DNA Artificial SequenceSynthetic 292 tgggcctttg attgcttgat gactgaa 27 293 18 DNA ArtificialSequence Synthetic 293 gtgggctcag cctttcca 18 294 20 DNA ArtificialSequence Synthetic 294 tcaagtcctt ccacacccaa 20 295 1424 DNA Homosapiens CDS (148)...(1119) 295 aggagcctta ggaggtacgg ggagctcgcaaatactcctt ttggtttatt cttaccacct 60 tgcttctgtg ttccttggga atgctgctgtgcttatgcat ctggtctctt tttggagcta 120 cagtggacag gcatttgtga cagcact atggga ctg agt aac att ctc ttt gtg 174 Met Gly Leu Ser Asn Ile Leu Phe Val1 5 atg gcc ttc ctg ctc tct ggt gct gct cct ctg aag att caa gct tat 222Met Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr 10 15 2025 ttc aat gag act gca gac ctg cca tgc caa ttt gca aac tct caa aac 270Phe Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn 30 35 40caa agc ctg agt gag cta gta gta ttt tgg cag gac cag gaa aac ttg 318 GlnSer Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu 45 50 55 gttctg aat gag gta tac tta ggc aaa gag aaa ttt gac agt gtt cat 366 Val LeuAsn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His 60 65 70 tcc aagtat atg ggc cgc aca agt ttt gat tcg gac agt tgg acc ctg 414 Ser Lys TyrMet Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu 75 80 85 aga ctt cacaat ctt cag atc aag gac aag ggc ttg tat caa tgt atc 462 Arg Leu His AsnLeu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile 90 95 100 105 atc catcac aaa aag ccc aca gga atg att cgc atc cac cag atg aat 510 Ile His HisLys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn 110 115 120 tct gaactg tca gtg ctt gct aac ttc agt caa cct gaa ata gta cca 558 Ser Glu LeuSer Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro 125 130 135 att tctaat ata aca gaa aat gtg tac ata aat ttg acc tgc tca tct 606 Ile Ser AsnIle Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser 140 145 150 ata cacggt tac cca gaa cct aag aag atg agt gtt ttg cta aga acc 654 Ile His GlyTyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr 155 160 165 aag aattca act atc gag tat gat ggt att atg cag aaa tct caa gat 702 Lys Asn SerThr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp 170 175 180 185 aatgtc aca gaa ctg tac gac gtt tcc atc agc ttg tct gtt tca ttc 750 Asn ValThr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe 190 195 200 cctgat gtt acg agc aat atg acc atc ttc tgt att ctg gaa act gac 798 Pro AspVal Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp 205 210 215 aagacg cgg ctt tta tct tca cct ttc tct ata gag ctt gag gac cct 846 Lys ThrArg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro 220 225 230 cagcct ccc cca gac cac att cct tgg att aca gct gta ctt cca aca 894 Gln ProPro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr 235 240 245 gttatt ata tgt gtg atg gtt ttc tgt cta att cta tgg aaa tgg aag 942 Val IleIle Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys 250 255 260 265aag aag aag cgg cct cgc aac tct tat aaa tgt gga acc aac aca atg 990 LysLys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met 270 275 280gag agg gaa gag agt gaa cag acc aag aaa aga gaa aaa atc cat ata 1038 GluArg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile 285 290 295cct gaa aga tct gat gaa gcc cag cgt gtt ttt aaa agt tcg aag aca 1086 ProGlu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr 300 305 310tct tca tgc gac aaa agt gat aca tgt ttt taa ttaaagagta aagcccatac 1139Ser Ser Cys Asp Lys Ser Asp Thr Cys Phe * 315 320 aagtattcat tttttctaccctttcctttg taagttcctg ggcaaccttt ttgatttctt 1199 ccagaaggca aaaagacattaccatgagta ataagggggc tccaggactc cctctaagtg 1259 gaatagcctc cctgtaactccagctctgct ccgtatgcca agaggagact ttaattctct 1319 tactgcttct tttcacttcagagcacactt atgggccaag cccagcttaa tggctcatga 1379 cctggaaata aaatttaggaccaataaaaa aaaaaaaaaa aaaaa 1424 296 2781 DNA Homo sapiens CDS(117)...(1106) 296 ggaaggcttg cacagggtga aagctttgct tctctgctgctgtaacaggg actagcacag 60 acacacggat gagtggggtc atttccagat attaggtcacagcagaagca gccaaa atg 119 Met 1 gat ccc cag tgc act atg gga ctg agt aacatt ctc ttt gtg atg gcc 167 Asp Pro Gln Cys Thr Met Gly Leu Ser Asn IleLeu Phe Val Met Ala 5 10 15 ttc ctg ctc tct ggt gct gct cct ctg aag attcaa gct tat ttc aat 215 Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile GlnAla Tyr Phe Asn 20 25 30 gag act gca gac ctg cca tgc caa ttt gca aac tctcaa aac caa agc 263 Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser GlnAsn Gln Ser 35 40 45 ctg agt gag cta gta gta ttt tgg cag gac cag gaa aacttg gtt ctg 311 Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn LeuVal Leu 50 55 60 65 aat gag gta tac tta ggc aaa gag aaa ttt gac agt gttcat tcc aag 359 Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val HisSer Lys 70 75 80 tat atg ggc cgc aca agt ttt gat tcg gac agt tgg acc ctgaga ctt 407 Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu ArgLeu 85 90 95 cac aat ctt cag atc aag gac aag ggc ttg tat caa tgt atc atccat 455 His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His100 105 110 cac aaa aag ccc aca gga atg att cgc atc cac cag atg aat tctgaa 503 His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser Glu115 120 125 ctg tca gtg ctt gct aac ttc agt caa cct gaa ata gta cca atttct 551 Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser130 135 140 145 aat ata aca gaa aat gtg tac ata aat ttg acc tgc tca tctata cac 599 Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser IleHis 150 155 160 ggt tac cca gaa cct aag aag atg agt gtt ttg cta aga accaag aat 647 Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr LysAsn 165 170 175 tca act atc gag tat gat ggt att atg cag aaa tct caa gataat gtc 695 Ser Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp AsnVal 180 185 190 aca gaa ctg tac gac gtt tcc atc agc ttg tct gtt tca ttccct gat 743 Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe ProAsp 195 200 205 gtt acg agc aat atg acc atc ttc tgt att ctg gaa act gacaag acg 791 Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp LysThr 210 215 220 225 cgg ctt tta tct tca cct ttc tct ata gag ctt gag gaccct cag cct 839 Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp ProGln Pro 230 235 240 ccc cca gac cac att cct tgg att aca gct gta ctt ccaaca gtt att 887 Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro ThrVal Ile 245 250 255 ata tgt gtg atg gtt ttc tgt cta att cta tgg aaa tggaag aag aag 935 Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp LysLys Lys 260 265 270 aag cgg cct cgc aac tct tat aaa tgt gga acc aac acaatg gag agg 983 Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr MetGlu Arg 275 280 285 gaa gag agt gaa cag acc aag aaa aga gaa aaa atc catata cct gaa 1031 Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His IlePro Glu 290 295 300 305 aga tct gat gaa acc cag cgt gtt ttt aaa agt tcgaag aca tct tca 1079 Arg Ser Asp Glu Thr Gln Arg Val Phe Lys Ser Ser LysThr Ser Ser 310 315 320 tgc gac aaa agt gat aca tgt ttt taa ttaaagagtaaagcccatac 1126 Cys Asp Lys Ser Asp Thr Cys Phe * 325 aagtattcattttttctacc ctttcctttg taagttcctg ggcaaccttt ttgatttctt 1186 ccagaaggcaaaaagacatt accatgagta ataagggggc tccaggactc cctctaagtg 1246 gaatagcctccctgtaactc cagctctgct ccgtatgcca agaggagact ttaattctct 1306 tactgcttcttttcacttca gagcacactt atgggccaag cccagcttaa tggctcatga 1366 cctggaaataaaatttagga ccaatacctc ctccagatca gattcttctc ttaatttcat 1426 agattgtgtttttttttaaa tagacctctc aatttctgga aaactgcctt ttatctgccc 1486 agaattctaagctggtgccc cactgaatct tgtgtacctg tgactaaaca actacctcct 1546 cagtctgggtgggacttatg tatttatgac cttatagtgt taatatcttg aaacatagag 1606 atctatgtactgtaatagtg tgattactat gctctagaga aaagtctacc cctgctaagg 1666 agttctcatccctctgtcag ggtcagtaag gaaaacggtg gcctagggta caggcaacaa 1726 tgagcagaccaacctaaatt tggggaaatt aggagaggca gagatagaac ctggagccac 1786 ttctatctgggctgttgcta atattgagga ggcttgcccc acccaacaag ccatagtgga 1846 gagaactgaataaacaggaa aatgccagag cttgtgaacc ctgtttctct tgaagaactg 1906 actagtgagatggcctgggg aagctgtgaa agaaccaaaa gagatcacaa tactcaaaag 1966 agagagagagagaaaaaaga gagatcttga tccacagaaa tacatgaaat gtctggtctg 2026 tccaccccatcaacaagtct tgaaacaagc aacagatgga tagtctgtcc aaatggacat 2086 aagacagacagcagtttccc tggtggtcag ggaggggttt tggtgatacc caagttattg 2146 ggatgtcatcttcctggaag cagagctggg gagggagagc catcaccttg ataatgggat 2206 gaatggaaggaggcttagga ctttccactc ctggctgaga gaggaagagc tgcaacggaa 2266 ttaggaagaccaagacacag atcacccggg gcttacttag cctacagatg tcctacggga 2326 acgtgggctggcccagcata gggctagcaa atttgagttg gatgattgtt tttgctcaag 2386 gcaaccagaggaaacttgca tacagagaca gatatactgg gagaaatgac tttgaaaacc 2446 tggctctaaggtgggatcac taagggatgg ggcagtctct gcccaaacat aaagagaact 2506 ctggggagcctgagccacaa aaatgttcct ttattttatg taaaccctca agggttatag 2566 actgccatgctagacaagct tgtccatgta atattcccat gtttttaccc tgcccctgcc 2626 ttgattagactcctagcacc tggctagttt ctaacatgtt ttgtgcagca cagtttttaa 2686 taaatgcttgttacattcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2746 aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaa 2781 297 68001 DNA Homo sapiens 297gctgtcttgg tggtggtatt tctgttgcag ttgttgtttt cttgcctgct tggtgacata 60tttctattga cttgacactt aactggcatc ttatctaggt agataatgct aattcaaaat 120tctgcagata ttgttctgtt gttttttgcc atttagggtt gagtaagatg ccaagttgtt 180ttttgtttct ctgtagtcat tctgttttca ttttgttttt agctttgcct ttggaattta 240aaatgttcaa aatgatttgt ctggatgaga atcgattttc ataacttttg ctttgataca 300ctaaacagtt tgagtttcta gatgatgccc attttaattc atacgaggaa atatcttcta 360gtatagtttc tgcttgatta attctatgtt tgtctcttag ggacatctat taattttata 420atgctgcctt tttttcagac ttctgtttca gaatattcgc tttcatcaat gtaatccttg 480gctatagtag gaatgaaata ataaaagcag tagcttctgt ctgccctcct tggttatgca 540gtccttacag aacatctccc catctcccat ccccccaccc cagctcagtg aaactctcca 600cactttggtt gtggaaattg gcagggttag gtggctactc actcccaatc cacatccaca 660ataaatcact ttttattatc ttatcaaaat ctgtagaatg cctctttatt ctattttgtt 720gctgcggagg tttgttttct ctttctaatt attttatttt ctaggttttt tgagggaatt 780tcaagagggg agatttttta ttcaggctca tcttaacgtc atgtctggaa ctcaagctac 840tgaattatat attctttaat acatatagac ctacgtcaat gagtttaaac tgcaaggaaa 900gggttaaatt tcttcctcaa gtgtggtcaa aatctgtaga gaaaagagga acagcttctc 960ttaaagaaag ttagctgggt aggtatacag tcattgccga ggaaggcttg cacagggtga 1020aagctttgct tctctgctgc tgtaacaggg actagcacag acacacggat gagtggggtc 1080atttccagat attaggtcac agcagaagca gccaaaatgg atccccagtg gtgagtaata 1140attcttattc tttgcagaga agttatgagt tgtgactgca gtgaaaggct gaggttgaag 1200atggtgcttt gatgtgtgtc cttcacttag ttcctaagtg gagaagcttt ctttttctac 1260aaaagatctt tggcacataa aggcaagaat tatttgcaat gcccaaagca gttcattggt 1320ggtagttata tatattttta ggtgcctaat tttggttttg taaatctgtt attcaaatac 1380tgaatgttac agtcattgat tttagtgaag aatcaggaat ttttaaaata tctgcataag 1440aatgacaaat aacagggaat atgttttttg tctaccaggg tcagtttggt ctgagggtgg 1500aggaatgaga tagagaaggt agagggagag agatcaagaa aaagaaagag aaaaaagagg 1560tataaggaga aaatgcaaaa ctcagttaat atgtcataat caggccatgg gagattctgg 1620gcagggttgg gtagtggaag gaggtagagt gattaaatta gttaccatgt attgaacatg 1680catgatgtgc tgggtacttt actagtgcta tttcattgaa ttttattctt cacaatgact 1740tttggaaaga catcatcatt ctttttgaca gatggggtaa ctgtggctta aaaaacttgc 1800ccaagttcac actattcata aggggtagag ctaaaatctt tcctgcccgc ttcgtggtgc 1860gccagaaggt ttctccatgc tgtggagact tcctggaagg agtcacaccc gcccttctct 1920tgggtggtgg cagctggcgc cagtcactat gtatttattt atttttaatt atttattttt 1980gaaacagagt ctcgctctgt cgccaagctg gagtgcagtg gcgcaatctc agctcactgc 2040aacctccgcc tctcggattc aaacgattct ccttcctcag tctcctgagt agctgggact 2100acaggcgccc accaccacgc ccggctaatt tttgtatttt tggtagagac ggggtccacc 2160atgttggcca ggatggtctc gatctcttga ccttgtgatc tggccacctc ggcctcccaa 2220agtgctggga ttacaggcgt gagccaccgc gcacccggcc gtcactatgt atttataatt 2280actgttcttt gaaaatcgaa gtaactttca tctacccagt gcttactggt ttgagaaaaa 2340gctttgttgc ttttatttca gaagattaaa atttaatttt ccagtaaaga ttccttttgc 2400tccagtggaa ttttgaagcg ttatacttgt atgaagaaaa aaagaatttc aaaatttata 2460atttttgtgg taccatagag gggatactac ttaattatgc tagcactgtc tgcagaggtc 2520taaaaaacca taggctgctg tctatattga acttgttaag attccttttg tttcacagtg 2580cctgaagatt ggtcatgaac cagtaatagc cattaaacaa tgtctgttct cataagagat 2640gaaataaata caaattaaaa caacagtgaa gtatcatttt ttctctatca aagagataaa 2700tattaagttt taaaaagcaa gcaatcaaga accctctatc ttgctaagaa gggaggatta 2760tttgtaccct agttgcgcac tagtggtgat atcatctttc tggacaataa tctagtgata 2820catatcaaaa gcctttaaaa tgtatatgcc ctttaaccaa gcaattcacc ttttaggaat 2880ttattctaag atataataat acatgtttgt aaagttttag tgatggatat ttttcttgct 2940attgtttcta ataaggaaaa tcttagaaac aatttacgtg tttaaaaaca agtgattgga 3000tgattatgga acatccataa tggaatacca tgtaattatt taaaattcca ctgtagaaca 3060attcataatg tgtttagtta aaggggaaaa atgcagaacg aacagtgcta tcacttttat 3120acatgataca tgtatacaaa caatattaat cagaaatata ggtagtttgt attttctact 3180gtgtgctttt caatttgaat cccatctgta ggcagaaaaa taaagttaaa tatttaagat 3240ttaaaaaaaa caatagctgg tttttttcag agaagtaacc atctagtgat gtgtgataat 3300ttataagttg tgagattcca tagttagggc tttagccctt tgcatttatc tttcttcatc 3360tcttgaatcc tcttcaaaat acacccactc tacccataac tcattagatc ttgaaaggca 3420tgttctgata gaattttata tttagaacag gctgcagcac tcttccctta ttttacagaa 3480gtgattgcat ggctctctag ggtgagttgc atattgagag ggaggacagt gcagtggcta 3540agtggccgca cattgaagcc aggctgctgg ggtttgaata ccagttcccc aacttcctag 3600ctatgtgatt ttggacaagt tgcttaacca ttgtaattcc cagtttcttg tctgtaaaat 3660gggagtatgg taatatgtcg taaagtagtt gtgagatttt aatgagataa tccatatcct 3720gctaagtact caggaattgt tagtagtttt tattactatt actgtttgga ttaagaaaca 3780gaggaaaagt gatttgtcca agattataca accacttaat ggcattacta agaacagaat 3840ggaggaaggt tttttccagc agaaatgttc agtatcctct gtgcctggca ggacaacccc 3900aagttgtgct tttgggatgg aggagctgat ctaaaacaag cagtacccag gacaaggcca 3960gcctccaggg agtgactgat gacagtggga agccaaatgg tagaaaggca ggtgaagtta 4020aggaaactga gagtcaactt aggagcagga atgaagcctg gagcaaagag ctagtgcaag 4080agagagcaga tgactcagag ctactggggc ttttgtaggc caccagctat gggcttcacg 4140ggaggttaat gtggtttaaa gtctcaagac ctgggtaatt aaatagacaa atggggtcta 4200ggtgcatggg ggaattttaa gtatagcttt gagaagatgc ctaatgggga gtaataatag 4260agaaagaagc tgggtggggc cagaacaagg agcccacagt ccaggcatcc agctatgatc 4320atcctaaagg aacagcctaa gtttgaagaa tcataaacaa atagagacat gataaaattt 4380actttaaaaa aaatcccttg gcagaagaat gaaaactgat ttggagtgac aaggagatca 4440tttaggagac tattggagta agccaaggag aaatggcgag ggcatgaaac cagggcagtt 4500atggtgggat cagactggag aggatgcatt taagagatat tttagcacca gaattgacag 4560aatttggttt ttgacagatg tagacactgg gagaggagga gagatctaag tgcatagtgg 4620catccttcac aaaatggaag gtataggaag cagagtgggt ttgcgccagg cagagagaaa 4680agacagatgg tgaattcctt ctctaacatg ttgagtttga ggtgcctgtg gagcagctgg 4740atagagatgt ccaagcagac aagtagatat ttaggtgcaa gttcaaaaaa gagggatggc 4800ctggaatgca catgaagagt cttctgcata agtatggttg acagttgaaa ttctcattgt 4860gggtcaattc agtagcagag aggttggagg attgagagga agccaaggac agaacctgga 4920aaaccttgac atctaaggag ggagatgagg aagaagaatc tacaatagat actaaggagg 4980ggctagagag actggagcag cccaggagaa aagtggtgtc atagaaatca agtgggcctg 5040tagtcccagc tacttgggag actgaggcac gagaatagct tgaacccagg aggcagaggt 5100tgcagtgagc tgagatagca ccacggcact ccagcctggg tgacagagtg agactccgtc 5160tcaaaaaaaa aaaaaaaaga aagaaatcaa gtggggagat ggatgcaaga aagaggagaa 5220tgcattcatg gaaagagcat atttaccaag ctttccatgt tgaacacatg agatgtgaca 5280tgaaaggtaa ctgtagtgac tacatgttaa gcgttgaatt gtggctccct taaaattcat 5340atgttgaaat cctaactccc agggcttcag gatttgatca tatttggaga tagggtcttt 5400acagagataa taaaatttaa atgaggtcat tagggtgggt cctaatccaa gacaattgtt 5460gtgcttattg taaggaaggg aaacacacag cggaaaagcg gtgtgaagac acagggcgaa 5520gacggccatc cacatacaag ccagagagaa aggctcgcaa cagattcttt cctcacaacc 5580ttcagaaaga accaaccctg tggaaaccct aagtttggac tcctggcttc cagaaaaaaa 5640ataaattttt tttgtttaag ccaccccagt ttgtggtact ttgttaccac agccccagca 5700aactaataca cttggtgaga gtgcatacag ccagagaaag aagctgtgaa taagtgcatc 5760agggaggagg gaaagaaacc aaaacaggat tacatcactt aaattaagag tagaaacatt 5820tcacaaagca gagtgtagtc acaggtcaaa ttctgcagaa aggcgaagta ggagaatgac 5880tgaaaatgtc agtccagagg ccctcagtga ccttgacctt tgcagaacac tcccagttga 5940gtggaggcag tagactttgg agagcttgga aaatggaggc agcacagatg gtctcctgca 6000gaaagtctgg tgataaaatg agacctcctc actggagtaa actcacctct gctgtcggtg 6060gaaataatct ggagtcaggc cagccagacc cagagttctt ccttcctcca ttttaaaggt 6120taaacagagc tgaggtcaat ggctcatgcc tgtaatccca gtgactcagg aggcggaggt 6180gggaggatgg cttgaggcga ggagttccgt tcaagaccag tctgagcaac atagcgagac 6240tcccattcct aaaaaaattt taatttaaat taaaaaaaaa ggctaaccaa aaataaaatc 6300caatacttta tttttcccac ccaaaactag tttgggaagg atttctggaa gaaaataatt 6360tttgcagtca ttttacatgt tggattttga gtgcacataa catacagatt ctattctgta 6420ttatcagttc agaggcaagt tgagatttga ggcttcgcag aggtaaagcc tctggtgaat 6480ctggtgagat aaagaagaaa acaagcccaa gaggaatttc agggatcatt tataatttac 6540atcaataaac agaatgggaa aaaaaaccca ttagagtttg gaatagagaa gtattaaaac 6600actttcttag aaagctttga gtcaaaatta atctttctgt agtggcagga atatgataag 6660ccaaacaacc ctaatgtcac agctctatat tattaggtgt cgaatcagat ttgcactaaa 6720acatcaagta aaaataaaag gaatgaacat ttggttaagt gaaccaatta gtcaatacac 6780gccagaaaat ggtaaaactg gataaaccta aaatactcaa ctacctagat taatcaaggc 6840caacctagat tatcacccca atattacaac tattttcaac caactaaaca ataaatcttt 6900atcaagagcc tgatagttta aggtactgtg atgaatacaa atgaaattgc tgatactttt 6960tttcaagtct atttagaaat agaaacccac aattatgaaa tgacaaaaac aattaatgca 7020gttaataatt cagtaacttt taaaaagaaa taaacatgac aaaaagttca ttctcaccaa 7080atactaaaga aatgccaatt caaacaccaa tgagatattt tcctgtatca gattagacag 7140taaaacaaac aatccaatca gaaaaatggg caaaagatat aaaaagacat ttccccaaag 7200aaaatataca gatggtgaac aaccatataa gagagtcaac atcatttgcc tttatgaaaa 7260aattaaacca ctacctacct ataaaaatgg ttaaaataat aaaagataat gacaacacca 7320aatgatggca ggatgcggag aaactggatc atgcatacat tgcttgttgg gaatgtacaa 7380tggtcaagcc actctagaaa acagtttggc agtttcttat aaaaccaaac atgcatttag 7440tatatgaccc agcaactgca ttcttgggtt ttgatcccag agaaataaaa gcctatgctc 7500ctgcaaaaat cagtatatga atatttatac cagctttatt cataatagta aaaaactggg 7560gaaaaaagtc cctcagtggg tgaatcgtaa cacaaactgt gtgtgcaaga tgttaccact 7620gaaggaagct gggtgaaggt acacaggact tccctgtaca ttttttcaac ttcttttgaa 7680tcaataatta tttaaaaatg aaaagtttaa aaagtaaaaa aaaaaaacaa aaactaaaaa 7740tgttcatctt cactaaatat taaaaaaaat gccaattcaa acacaagata ttctcctttt 7800acaaattaac aatttatatg gattttggga gggttgggag taatcatgct aataagtatg 7860caataagagg atactttcgt atactactga ttgtgggaat atgaatgggg agaacatttc 7920tggaaagcaa tatgtcaaca atatcaagag tcttaaaaat ggttgtacaa gcagacaccc 7980attctgggca ctgccaattt ctccatgtcc ttagtacatt ttttttcagt tcattcagca 8040tctttgttcc aggcactgtg ctaaacatta aaaatacacc aaagatgagt atgagtaaac 8100atgatttctg ttctcaagaa tttcagtttt gtggtaaata tatcaaaggt gattttttat 8160aagagttttt tataacaggg tgtgacgttt cataggagca tgaaggtagc tgtttcctat 8220ttgtctgtag gcagtatgat gtcttagata aatgccaggg ttttgagcta gtttggttgg 8280tatcaaataa taagtagtta ataaatcatc ttctatttat tagtggtatc actttgggaa 8340gcctattagc ttcctgaact tcagtgtcct ctgtaagatg aggctactaa gcacttgcca 8400atgccatgag gaatataaca atttataatg gacaggaagt cctatggata taagatattt 8460taggactcac attctttgct ttaaaatcta ttatttccta tatttttaat tgtcagagtt 8520ctttagctct gccttttctg attgatttcc agcagatgga ctcttaccta taacctagaa 8580gttgctatag tagacctcct aactatagat aagagaaggg catgccaaat gcagttgaat 8640caggtgaaag tcaagcaaca aagctgccta aaataaattt tatgtaaggt agggtgccaa 8700aatcattaaa ataaaattct attctataac tgtaatcacg taagtgcttt catgaagttg 8760tctatgaaaa ctttcttttt cgctttctgg acttcaaata ttttaagttt gcttttcatt 8820tacaaagatt tttttgctca ttagtaatca tgaactgtat tcaaacttac acttctaatt 8880ctagaagata tataaactac cattttttaa ttataaaaat gtttatatat cttgctttaa 8940taatttcacc tctagggatc tagctagtta aataacaaga gctacggaaa catatttgtg 9000caccaaaatg tttattacat tatttaatgt aatgaaaaat aagaatcaac ctaaataagt 9060agaagaatag gtaagtaagt taaaaatgaa gttaataatg actccttaat gagagaagac 9120aacataaggc tacatacaga attgtaaaga aaataatcca cagaatgtgt gttttatttt 9180ggtaaatggt tcctaaaact aagtaagtac atagaaagat tttttttttt ttttttttaa 9240agacagagtc tcactcacgt tgtcacctag gctggagtgc agtagtgcaa tctcagttca 9300ctgcagcctt cacctcccgg gttcaggaca cgccaccaca cccagctaat tttcttgtat 9360ttttagtaga gacacagttt catcatgggg ctggtctcaa actcctgacc tcaagtgatc 9420tgcccacctt ggccttctaa agtgctgtat tataggtgtg acccaccgca cctggcctag 9480gaagaatttt agaaagaaag ctccatatca ggaattgaga agccggtgtt ttaattgaga 9540atatatttgc acagaaaaat cttggcataa atattggttt acaaaacaaa caaacaaagt 9600aatgtccttt aatcttggat caggagctgc ccaacaactc caaaaagtca gctcatgcaa 9660aaccatccaa ggacagatga atcagccaaa caagagagaa aggggaaggg aaagtgtctt 9720ttcacaggca gcttttgagg cagtgcataa accatgcctc tgcaccatcc agaccagaca 9780gttgtgacac agggttgaca aagcaggaca acgaagggta gctgctccta aggtggggat 9840gatgctggag caagggggag caccaagagg aaaaaaaaaa agcataaaaa taagatagca 9900tagtaaaaaa taagatagtc atagtagtta cctcttgatt gatgggatta tggaaatagg 9960gttatttctt tgactaaaat tgccagatct tcagtacaat tacattgctc tgctgagcag 10020gatgaaatca agttgaaaag taatctagta gtgaggtaca gcccgtatgc tgcaaatggc 10080caacatagat cctcagatga cagaagtgag tgatgcaggc ctgtggttta cgtacagctc 10140catgacgtat gaatggcaga agctgtgtat gtccacaggc gagccccatt tcaagaagtg 10200cttctggtca ccactctgtt gtcctgtgta taaggatgtg gttcagaaca gccaagtctt 10260atatttcaag aggagccaga aagacaaact taccagtgaa atcctaatat ttatataata 10320gctcaatatc tggaggggct ggtttggtat aaatcaccag ttttacctct gacctctgtt 10380aatgcatcag aggttcagga gagagtgaga aaattgtaaa tggaatattt taggactatt 10440atattggggc ccaggcttta gtgagtcaga gcgagaaagt agggggctga aactatttga 10500ctattaattt attcaataaa gttttattta atgttggaaa gatgaagatg aatcagatag 10560aaatcctgtt tagaagcttc tatgggaaga gatatcacca tagctaatat gtcttagcct 10620ctaaaaggaa ttatggcaaa ctatattgta tcatatatga tctcatttaa tcatcataac 10680tgcaggaggt aagtagtatt atccctgatt ttccataagt gaaaactgag tctctcttag 10740ttacatagct gacaacacaa ccaggattcc aaatgccagt ctgatcccag agccaagcta 10800tgaacaacca tgctatatat tatggcagat cagggaagga agacattact tctagcagca 10860agaaacattt gtggatgaaa agatttgagc ttggagagag ctgtgttgag ccatgtccaa 10920aacaattttt ggcaaccata cgataaaggt aaaagccaga gattgaaaag taaaagctgg 10980tggactaaaa atggtccata gacagagagt catcaaaaca acaaaacaaa acaaaaacct 11040taatcataat taattgccaa tatttcaaaa gtctggagaa ttcacataag tttagatttc 11100cagctcttct ggagaattag aatatctagt aacagtgtgc taataatccc acatggtaac 11160aactggtgaa tctggcagag gctgcccagc ttccaatggt gtataagctc tcctcttcct 11220atgcaacctg cctgcctcat ttatatgagc tgcttggatg ctgcagatgc ttgaatctaa 11280gagctttggt ctggaaagtg aggccctaac aaggaagaag gaataaccta tggatcaagg 11340agaactggga gtccagaaat gaaggttatt tgtaagtctg gacccagcca ttccaactcc 11400tttgaggaaa taagattcta aggaaaaggc cgtttgcatt ctgctctcca agatctctgg 11460gtgttggaag aaactgaatt gggggagagg gggaaacttg actgggggct caatacagac 11520atgtaaattt gaaggaaaca gagagtttag atgacaggca gtagaaaagt taatgtgtcc 11580attctatggc tgacccaaga ttctgtttcc agaagacctc tctggcttgt taagtgttca 11640tggttgcagg ggaaaagtta gaaaaaaaga aaaacaagcc aaaacccagc tccttaaatg 11700tttctaattt tattttcaaa caatcaggca gagtaatccc tttacacact cttcaggcat 11760tggctgaggg tcccagtcaa gaaccattca gttttggggg ccttaagaaa aatatttcct 11820atgattaaag gaactttgga caggttatta ccttctttga gcctcagttt ctttgtcact 11880tagaaggttg aatggtttcc acattctgag ggtaaggaat acgagagtga atgaagaaat 11940atcaagtgca tagctcagag taggaagaaa gaagtgacca gggacaaggc taagaactta 12000ctcaaaaggt gcccagctgc tcagcattct gtccaaaaaa gggacactga catctctcca 12060gcattctaac agcagtcaca tagcattatc agctagaaat gaaaacagat tcaattctat 12120atcctgctaa aagcttgagg gtcacactag ctgtgtgatc tttggcagct ggccaaaacc 12180ctctgaaagg cagtttcctc acctataaat tttttaaaaa atatttattg tgaagattaa 12240atgaagtaat gcatttaaaa tacttagggt gccttagagt tccagcacag ttcgtagctc 12300atagtaatca gttaatagat attgacttta ataaatagat acttaactga gcctccaccc 12360tgggcctgtt actatgctaa gtaccaggag tgcaaaggga aatgaaacac gttccccaaa 12420cttgtggaac tcagagcagg ccaactatat gagtagtaga tttttaacac gatgaggaaa 12480ctgttataat ggaaaaataa atagtgtgaa gagggactaa ggaagataaa gatatggtga 12540aaaaaagagg gttcaggctc caactgtggc atgatctaga tctgcaagga agagtagaga 12600attgcagtag agaaagcagg gaagacattc caggcagaag aaacagcttc cacagaggta 12660aagaaggcaa aacgttttca aaggttctag gggtggggga tggtggggaa gatggtcacg 12720gaatagtgaa tggttcaagt gaaactgttg tatgggttgt gagggcagtt ttatgggttg 12780ggtgataaaa tcagaagaga aaaattgaat tcacgctttg aaaggccttg tgtgtcttgc 12840tgagaagcgt gaacactaac tcatgaggag taaggcgcgg gtctttaaag gaaggaagtt 12900tcataatcaa attgttttcc atgaaagata atgaagggag ccagccacaa ggctgttgca 12960atcaatcgtc caaacaagag gtgacaaaga ctctaaaact ggccaccaat gaattgaaag 13020tggatggtga ggaagaaaga atacagggtg acataaacac aatatgtaaa atccaaggta 13080tgaaaatggc ctggtccctt cgtccttacc acagtcatcc caagagacca aaacaaaaac 13140acagaaaatc tcttttaaaa ataatctctt ttgtttgtat gcaaataggc catccacagt 13200gaaaatgcaa ctcaaatgca atattttatc tgcagtccac caaatgcaaa gatcaaattg 13260gtttacaaat gctgtccttc ttaaaaattc caactcctca cattaaaact gtagccagct 13320agtgcaagtt aagattgttt gcaatcttaa ataagatttg agtaaagctg aaattgagac 13380acttttcaaa agaggccatc ccttactcac attgctgaag agtagaaaga ttgacacctt 13440cttttatcag aaaatttctt tcagggagta atgcctcttg tgtggtggca gacccttcaa 13500gtcttccaga taaagcatgt gatggaagta gcagggagct gcaaaaattg caactctatg 13560attctgcatc accgacttga aaactacaag cccaggttga caaatgtaca ttttaagtgt 13620tcagagaagt cttaagtgcc ttgctttggt cacaaagttg ccacagggaa gtaagttttt 13680gaatgtgcag tgccccgtcc ccagctctgt tgtgaaatgg aaactttaaa aaaaaaatca 13740ctgatttaaa aaaccactgg ttttgttttt taacaagttt agtcatattg cctgtgttct 13800atatacccca atctatttta ttttactttg tagtgtacat ttaatttata ctcaaataaa 13860tattttacat aaggtgcttt cacggacctt catgcttccc taaatattaa atatgctgcc 13920catttgttaa aatgtgttag ttttgttatg tattatatcc cttcccctgc acacatagaa 13980aaaaaaaata tgaacttaac cttcaagcaa gttttacgtt tagaatatta cctcatttta 14040tttcttctaa ctgaggttat aaagaaaatg gcaaatgtca tgtgctttgt aaagaaatga 14100cagtttctca gaatacgtaa atgcacaccc ttagacaaat aggccactta aattcagaat 14160cacagtcttt tgaatattgg tttaatattc tcagataaag attgaaagga taacaagttt 14220tggaatcagg tgtttacttt gagttctgaa aggtgacatc acaggtgttg gtagtttatt 14280gatatttaat ttttaaatgt gctgctgtat atatttcatt tgattagaaa tatgttacat 14340agttatgtta tttgttaaat aatagaccat cttttgtata acctatcaga agaagttctg 14400agattgtaag attagattgt aaatcctatt gcataactag aatacagaat tattaaattg 14460gaaaggaagt taaatcactt agaccgtcct tcccccagga agtcctctct acagtgttat 14520ccatgacaaa tgtttatgca ggcaccctaa ccatttaaaa tacgaagaga aggagaagag 14580atatatcgag aaaactattc ctgagacaag agaccagggg aaaacttttc ttagttgtaa 14640cctacacaac tagctagaat ctacctcatg ctcttaagat ggtgtttaat catgaggata 14700tcatacataa ctgaaattgt ataatgttga acatcttttg gtggcaagta ccttgacttt 14760attaaatcag tgtaggtctc ctgcattcaa ctatggtctc taggagggca gggaccacac 14820ttcttagctc accttggatc tcaagaccag tccaaggcct tcattcagtc agagctagtt 14880aagcatttgt taattgaatg aaggaaatta aaaaaaaaaa aactagaaat tccaaattgt 14940gcaattacat ctgtgaattt taaggtgtta ttggaaaatg ggaaaaatta ctgagattcg 15000aagatggata ggaacaaagg taaagcatga aaatgtgcat atttgtgtgt tgctagagtc 15060aatagtgatt gccagttcct atgcagccct ggcaccacct ctgaaacctt ggccaacccc 15120tgccattagg tgttgagata cacgacaggc aggtgaggaa ggaggggtgc tctggatgta 15180gtcttgccgc tagactgtgg tctttagtgt ccatgtccat ggggtgagtt gtgagcccat 15240cagtttccca cactacagtc cttctctggc ttagccttcc ttccctgtcc tgtggtccag 15300gttacccctg gcccctgtgg ccttcctcca gagatggacc ttcctccaca cacttctcag 15360gagctcctga ttctaggcat gccccagaac accgcaactc cactctgccc tgtctccatc 15420agaccagaga cctcacttag accctgggta tggggttgtt ggttctccac ttgctgcggt 15480tgtcactagc ctgagctcta tcctgagctc tgtccatcct tctcatcttc ctcattgccc 15540tttctgctaa agcaactgca catctgaagg ctatacttat ccctagtaca atggtacttt 15600ttctaaaatg catacaccct aatgcttacc cttttgacaa ttttttcctg aacctacctt 15660ttaatataag caaattcagt cttcaattca aataagtgta ttttgctgct gaagccacca 15720tgtgattttg agagatagtg aagacagaca gtcttctgca ttcacttgta gaatcctgaa 15780aatacctctt ggtgtccctt gcctttctga cttcttgctt gaagacatct agacaaaaat 15840gtgtccctgg gtcctagttt ctgggttcag aaattgatcg aatgcaagaa aacaatacac 15900attgcctctt ccttagcatg catgaatggt aggtgtgaat ttgcatcatg agaaagtaaa 15960taaaagaaga ttccccaggg cagctaggga ggcagaaaaa gccagcctag gatgggagtg 16020gaggacatgt tagaggttat gagagtgagg gtccatccta ccccacctcg gtaactcact 16080ggtatggtat aaatgcaaaa ttttggctca cacaaagaaa aatactcaac ttctaatgct 16140taactatgta aaatttgctt ttaaagtaca agttaaaatt gtatcgcccc tcaaagaaac 16200aagaaactca ttacatttct aagaatgttc cttcagaaac atggaactga aagctatttt 16260taaaaattga tctggccctt agaaaactgg ggccttttct ttaatttacc taaggaattg 16320acataaaagt ctagggttct gcaccagaaa aatgcagaaa gtgtcaaaat aaaaggcaga 16380aatacaaaag gagacttttt gcagcaacgt tctatgtata gcattgattc caagggtgca 16440acatagggaa gtgaacatgt ggactgtgaa attgatgcta attttctttc ccactagtct 16500agcagccctc taaaatgtca cattattaat ttagttactt taccagaaat ccgtgtatgt 16560ggttagcatg tgtgtttttt tttaattaac agactttact tatttttaga acagttttag 16620attttcaaaa aattgagcac atagtacgag aattcccatc tactcccttt gtggaacaca 16680atttccccta tttttaccat cttgcattag tgtgatgtat ttcttacgat caagccattg 16740ttgcttcatt attattatta tttaaagccc ataatttaca ttaagtttct ctctttgggt 16800tgtacagttc tatggatttt gacaaataca caatgtcatg tatccaccat tatagtatca 16860tacagaatag cttcactgcc ctaagaatcc cctgtgctct gcctgttgac ccttcccacc 16920cctccccaac ccctggaaac cactgatctt tttactgtct ccacagtttt gccttttcca 16980gaatgttcta tctttggaat catacagtat gtaccctttt cagattgact tctttcatta 17040agcaatatga atttaagttt tctccatgtc ttttcacatc ttgatggctc atttctattt 17100attaccacat aatattccat tgtctggata taccacagct ttaccaactg aggggcatct 17160tagttgctaa ttatgaataa agtggctata catattcacg tgtaggtttt gtgtggacat 17220aagtcttcaa ttcaattgag taaatatacc tagaagtgtg actgctggat catatggtaa 17280gagtatattt acttttttaa gaaactgcta aactatatcc caaagtagtt ttaccatttt 17340gcattctttt ctttattttt tttttattat actttaagtt ttagggtatg tgtgcacaat 17400gtgcaggtta gttacatatg tatacatgtg ccatgttggt gtgctgcacc cattaactca 17460tcatttagca ttaggaggta aatctcctaa tgctatccct cccccctccc cccaccccac 17520aacagtcccc agagtgtgat gttccccttc ctgtgtccat gtgttctcat tgttcaactc 17580ctatctatga gtgagaacat gcggtgtttg gttttttgtc cttgcggtag tttactgaga 17640atgatgattt ccaatttcat ccatgtccct acaaaggaca tgaactcatc attttttatg 17700gctgcatagt attccatggt gtatatgtgc catattttct taatccagtc tatcattgtt 17760ggacatttgg gttggttcca agtctttgct attgtgaata gtgccacaat aaacatacgt 17820gtgcatgtgt ctttatagca gcatgattta tagtcctttg ggtatatacc cagtaatggg 17880atggctgggt caaatggtat ttctagttct agatccctga ggaatcgcca cactgacttc 17940cacaatggtt gaactagttt acagtcccac caacagtgta aaagtgttcc tatttctcca 18000cattctctcc agcacctgtt gtttcctgac tttttaatga ttgccattct aactggtgtg 18060agatggtatc tcattgtggt tttgatttgc atttgtctga tggccagtga tggtgagcat 18120tttttcatgt gtcttttggc tgcataaatg tcttcttttg agaagtgtct gttcatatcc 18180cttgcccact ttttgatggg gttgtttgtt tttttcttgt aaatttgttt gagttcattg 18240tagattctgg atattagccc tttgtcagat gagtaggttg caaaaatttt ttcccatttt 18300gtaggttgcc tgttcactct gatggtattt tcttttgctg tgcagaagct ctttagttta 18360attagatccc atttgtcaat tttggctttg gttgccattg cttttggtgt tttagacatg 18420aagtccttgc ccatgcctat gtcctgaatg gtaatgtcta ggttttcttc tagggttttt 18480atggttttag gtctaatgtt taagtcttta atccatcttg aattgatttt tgtataaggt 18540gtaaggaagg gatccagttt cagctttctg catatggcta gccagttttc ccagcaccat 18600ttattaaaca gggaatcctt tccccattgc ttgtttttct caggtttgtc aaagatcaga 18660tagttgtaga tatgcggcct tatttctgag ggctctgtca ctatacatct actagaatgg 18720gtaaaatcca aaaatctgac aataccaaat gctgctgagg atgtggagca acaggaagcc 18780tcattgctcc acatcctcag cagcatttgg tattgtcaga tttttggatt ttagccattc 18840tactagatgt gtagtggtat cttactgttt taatttgcaa ttctctaatg aggtatgatg 18900ctgaccacct tttcatatgc ttatttgctg tccgtgtacc ttctttggtg aggtatatgt 18960tcagatcttt tgctccttat taaattgggc tgtttgttct tttatctttg agttataaga 19020gttcattgtg tattttggat accagccctt tatcagatat atcttttgca aatatttttt 19080ccccaatctt tggcgtgtct ttttattcat ataatggttg atacatgttt ctgctttaag 19140gaggaagggt tttaaaaata caatttacag tagcagtaaa aataaaaatt tattgcaaat 19200gtcttatgtt cactctcagg tgatgtcagg gaactatgga cccagcaggg tttaattaaa 19260ggggagtgtc aagtcctggg ggctgtggtt gacaatcctc ctttattggc aattgtgcag 19320cagggctggg agtaagaaga caacccagtc ctgagctgca tcacttctaa attaagaata 19380attcaggaac tgtgtttacg gtgaaatcct ggcccttctc acatagatta tattatgcat 19440aggatatgaa tttctgtcca tgaatccaag tatatatgaa atcatcactt tgaaaatttc 19500ctttaactca acttaatccc actggtgagc ctcaatcctg ccagttgaaa aagagactgt 19560aactgggtca tgcaggagtc tccttccttt cctgcagccc agtcagaatt caagaagttc 19620acctggtaac tggaaaatga tggaagggcc tcaagtccct agtctgtcct ggttgccatt 19680ggcaccctta ctatctgagc ccatagtggt ctgtgaagtc cggcagctcc ctgcccccat 19740gccacagtgg ggaatgagaa tatctactga tgctgggccc catgagcaaa gcatgctgcc 19800tttctaggca tgagccatca cacctgaggt tgctaccccc tcgggagcac tgatggaggg 19860gcagttgggt ttctactgct cacaggaccc agacaaccat cccctgccct cccttcttct 19920tgcacttcaa aagcactctc ttcctctctt ttcacctcta agccaccggt atcatctctt 19980ccatgggctt tcacaaaagt ctggatgaac ctttgaactt gtatctcttc tgctttcccc 20040cttgcatcaa gaaagcttag aaaacaaaca ctaattaacg tttcaataaa taatgctgct 20100ctaattattt gtggaaacta ttctgtatta gaactaccat cagcaccgcc tcctagagtg 20160cttttagact tgaccactgg ccgcaggagg ccacttccat ataacaacaa acagatggct 20220gaaattggaa aactcagcta aatgttcaga tgtttctaga ctcccacggg tttctggctc 20280tggcacatgg agtagatcct gactgtgtgg tcctcagggg actctctctg gtgaagtttg 20340gtgaggtcaa cttccacacc cacacacacc agctactgtg tgtagcctgt cctcctctgg 20400ttgcttctac ttgcagcctt ggcctcttca gtcctgagag cgttggagaa tgaggcagtg 20460gaggaagcag ccccacacag aaagcagttt ctgaagtaac ctcagcaact tcctcctcac 20520caaacacaag gaactgatct tctccactgg gctcggcctc tggtcagcca aggacaacac 20580tgttgaccac catcacggtt ggccccactc cacccttggc tctgatgaca tatgtgggag 20640tcagaggaat tttgattggc tgactgctgg cctgtcacac aaacaagatg gggcaagggg 20700gttgcgatat gacttgacat gtgaaaaaaa aaaaagccgt ggtcagcaac cccctgcaac 20760tgttgaaagg ctaattcaat ctctgactct ttaacaaaag tgatcttgtt cactgcctgt 20820tctgccctga gagccttctc tgctaggagg taggttgact gactcaggga gaagggtgct 20880ggtggcagag ctgccaatgg gtgagggtcc tagagactat cgacatgagg ggcagttgag 20940aacactgtag tatttagctg agaggagaga ctattaataa aatttacaaa atcagctttc 21000agctatttgg aagggtttat ataaaaggat aaaataatat gttctggtag ttctagaaaa 21060caggacagag acaagtagct ggtacttatg gattggagga gtgagtggca gtagtttggg 21120gattatttat aaaaaagaca tttttctgtt aactctcttt tctaatagtg aatttcccaa 21180cctgacaagt aagaaagcac aggctagaca cgcatctgtc atgacactga aggggtcctt 21240gcttgagtga gagactggaa tgatgagttt tgaggtccct tacagtccag caactctagc 21300taagttggag aataagagaa ttccatgaca ccatatcacc ccctcatttc tgctgcctgc 21360ctcaccattc atctctcttt actcctttta atatcattct acgttacagc attggaggag 21420gctgctctaa ataggaactg aaataagtag attaaagaag tgctatggaa gggaaaacaa 21480taaaacaact tgttttttaa gagcctacta ttgccaggat ctgtgctaag caccatatat 21540atgccatgtt atttaaccgt catgacatgc ctatgagata tttagtatta cttctgtgag 21600gaagccaaag ctcagagagg ttaaataact gccccaagaa cacacaggca ttaagtagtg 21660gagcagggtt tgaacacagg tctctatgac tccaaagtgc agtgtgatat gttattttta 21720ctgatctgtt tatggaaaat gatactgctt tctaatttag tattaacaca aagatttttt 21780tctaaataga tttacttaaa gtatgttata aaaatactat ataaataatg aaacagattt 21840tacatgagta tgaagtggta ctagtagcta gaatgatgaa agtttgggga atactactcc 21900aaatattttg atagctagcc tttcaattta gcctgtctta tatttggact gctgagtaca 21960aggaaaagaa ggaaacatga aaattaagtg aaatatgagt tacttcccct gtgctctgat 22020aggtgggtaa ttgatcatat gtcacaataa gaaaatcaaa tgaacccttt caaacaacag 22080caaaatctgt gattgtaaaa tccagaggaa aaccccaggt gggatctatc tgtatgaagg 22140atgaaatttc caaggtctga acatagaatg gctgagagga agtgatgacc ctgtgagtca 22200agaccctgga ccctggggga gccctgtggg tttgagaagc cctgggtgaa aggtgaaggg 22260ttttacaggc ctgtttacag acctctgtag tgacagaagg gagatctttg tgcaaaggtc 22320aaagtaagaa ttgggaaagt ctgaaaagaa aacaggaaag taataatgaa gatgaaataa 22380ctacttggca tactctgcca catgatttac aggcaaggtt tcctttgttt ttcacaacaa 22440tgcagcaaag aagtgattat gagtcacatt tcataagtga gaagactgac attcaaacat 22500gttcaataac tggcccaggg tccagtggtc aagccaggac tggacttcgg accaccagtt 22560ccaaacccac accccttccc ttgcaccaca cgcttttgtg tggatgagcc tccccaaccc 22620tgtcaacaac aaactgtcac tttgtcactt ttaatgtctc ctgcttcaca ggacacagct 22680agcctccaag agatcaggga ggcatgccca gagggtgctg cttctctctt ttgaagctca 22740agtgccacag acctcagagg cacataaatg tcccccacac tgagcagagg actttgcagt 22800gcctgatcag ggcagaaaaa ggaggcatgc acctggggga ggatcacata cgagtgaaac 22860ctgtccccgc tgaagcacta ggtttggaga aatctactgg gcatttacac acctttccca 22920cttctgctta tgacttgtag ccaaactcaa gagtaccacc cacttccagg aatagtgtac 22980caaggtaaca gaaacattct agattcatac aattggggtt agattaggat catctgaaaa 23040tgaaggttgt gtatgtcaat tgccttctaa caggatgggt ggagagatgt acttaatgaa 23100tgattttggg gaagggctag aagtgaagca catggcctct ctgccctcac tcattgaagg 23160ctgtcttctg aagccccgtg gagctcagtg cctgtcacat ggttgcccac atttgttgaa 23220ctgaactgca ttttcatcta tgggcttcaa aggctgtgtg tactctggga tctctgggaa 23280tctgtcaggg aaggtgtctt tgtcatgttt gtggatgggg ctccctttgg gggtttccca 23340gggctttaca ctcatgctcc gagggtacgt ttgtagtcat tctcatcagt ggaaatgccc 23400acctgccggc agaagttatt tggaaccaag caagagcact gtccctggct gtggtgttgt 23460ttctctagtc agttcccctt tctgtatttg agttctaccg tcagtcctgg cattatttct 23520ctctctacaa ggagccttag gaggtacggg gagctcgcaa atactccttt tggtttattc 23580ttaccacctt gcttctgtgt tccttgggaa tgctgctgtg cttatgcatc tggtctcttt 23640ttggagctac agtggacagg catttgtgac aggtatgttt gtggaggctc agacgcctag 23700ggagtggcat gagataaagc tgcaagctgc atctggggca gaaatgctga tgtgctaatg 23760gccggccaga gaatgagtaa aagggattgc agagagcatg cttaaaacct ctgaccatca 23820ggtttgcttc tcagattgac tacattggag gtgggatatt acaaaaatct gtctcttcct 23880gccagatccc ttcatctgtt tttcgtgagc taagagacaa aataggcagg aaatagaagg 23940tgccacttac caaataattg gcagctgttc ttggctttgg ggtgctgggg tctccgagca 24000gcctctgctc tagaagaagc agtccaaaga tgtcagctcg cctcgcctga gtcccctgtg 24060ccagtgggaa atccagagaa gggggatttc ctcctcttgc agcctctctg caatggactt 24120acttggcttt cctgtttgac ctttcccttc tctggtccag agacccttcc ccaatatttc 24180ttcccatcca agtgccccat cccaatatta gccccacttg gcaccagaga ccaagatcta 24240atttaaaaag aaatattctt gggtcaaaaa agagcccaag caagtgattg aacataatgt 24300gtttcacata cggtgaacct atttgcattt gcatttgcaa acgggcttaa aatatcatct 24360ctattaatag caatttaagg ttctggagag ccaggtgaaa atagtttttg acaaagggaa 24420cttcctactc cccttaaact gtaataatga aggaaatgaa ctgtttatct tacatgtaac 24480ctcaatcttg ggactaaggc cctgtactaa aatgcgtcta tttatgtgct cagacttgca 24540gttcgtgtta tgtctgctgc tgcagatacc gttaatatta tttatgtgag ctatcctgtg 24600tataatggaa gcttttataa atctctattt atttattcct aatatagtta ttaagtgctt 24660gctatgttcc aggtactagg gacttaacag gtagcataaa agacataagg aaaagctgca 24720ctcttgtttt ctagcctagt ggggaaatca cattaattta atcacactaa acatgactac 24780atagcaatag tgctttaaag ggaaggaaat tgttctatgt gactatatca gctgattaat 24840taccaagcct ttgcatttga tattttggtt agtctattct tcttgaattt catatgcctc 24900ttcctgggtg ggggtgagga tgggatttta tggagttgag gctagggcag gtagggagaa 24960aacatgagaa agatgaagag ataagccaag ccagattctt cagcagaaaa atcaaggttg 25020aaataccatg tttcaaaaat cagactgagg tgggagttga ggttaggggt ccctaggcca 25080ggggattgaa gcttcaaaga gataaaacta gagcaaaagc aagcacagag agtggcagag 25140aggtccctgg gcatttttcc acagtccatt ctagtgctgg caatccacct ttcatggcca 25200ggcaggtaag agtatttgtg gggtgggaga aaggacaggg ccataggctg ggcacacagc 25260cctttactgg cccttatctc tcctctcttc tcctatacag tgctgtttcc gaactgtaca 25320ttggcttaca ctcgggctga ggtttgggaa ataggcgcca ttttgaatat gtgtggagga 25380agaaaagtgt gtcttcagca ctttccacct ccccatcacg gccctgagac ctcaacaccg 25440ggaagcatct cgttccctat cggtcctcct ttattcatgg acggatatga ttcctttcta 25500agttccatgt cctttttaga taaattaact tgaacctaat gcctaatggc ttaaaaacaa 25560acaaaaaaaa ccctcttcct tccagctagc atttgcattt taacaggggc tttcaaaaaa 25620tgccttagcc caaggaatga gtaatgtggg aattccaagc agcagggtag gactggtgca 25680cagtatgggg agagaaggcc cctcaagttg tggccctgaa atgttggctt cctctctttg 25740accatgatgc tgtttctgag aaaacaagaa tcaggctacc ttaggggacc aggatgggca 25800tggctccctt ttagtgagtt ctatgagcct catacctgac agtcagagcc ctcgagtgga 25860tgagcacaga ctagaagaag cactgtgaaa ctttgcatga tccttacctt tttggcaaaa 25920aggaaaaaaa atcgttctca aattcatcaa tagtttgaaa tagggtgtgc cttgattcag 25980aaagtttcga ttctagatac aactcggaga actaggcgtg tcttgtacac agatttgctc 26040ttgggggacc ggaaaagcta aatgctatcg ccatgctatg ctccttcttc taggccagtg 26100aggggaacgc attcttcatt ttaatatttc agttgcctac aatattggaa ggtggataaa 26160agcaccctct gctccttcta aatctgcgaa gacatttctt ctctgcacct actcatcctt 26220gatgcagctc tcctcatgtc tgtatggaaa cactgtgctc tcaaatgagt ttcagaaaga 26280acaactcacg aaagaaaaca agcattcggt cagaaaaatc tccacaaatg gggaataagg 26340gggatttgct ccaaggagag actggaaacc aagtcagaca taaaatccag cctaagctag 26400aaggagacat ggctggtggg agcttgagga aaacagagct caggatggag gacgtctcca 26460cctccagtca tgtcctctgt ccaccagaca ccaagaagtg ttcatgttcc atcgaggcag 26520ccctcacacc catcccttcc tcatcatgcc gactgcctct ttactgcttc aggctcacca 26580tctcaagtcg acgagcctgt aatactggct ttcttgatca ccctgatacc agccgtcacc 26640tcttgacagg cttattttct ttaagctgtc attacaccat ttttctgctc ccaaactatt 26700aattccaaac ttccaatttt ctgttaaatt aaatatgaat tccttatttg actttccatg 26760ccctattagg ctatcttgct ccttgcttta cttatagaaa ctaatctccc attatttatc 26820caaagacaac ctctgctgca ggccagtcag cttttcttac tgtcctgtaa aaattccatg 26880gtcactcctc catttccatg tgtccttaaa aactgttatt tgattgtgtc tcagaaagtc 26940gtcaaagaat atataccaat gaaaagcatc aaaaaggtta tacttgatgt tatgtgtgta 27000tcaaaaatat ggctgaaata tttatccagt gaaactcaat caacactaaa aagtggttct 27060ttcggaagca tcagttcttt gagacccatt aaacagatgc ctcggatgca gggttatata 27120ttatcaggaa tctgtctagg gaagaattat tggaagcttg caaagccttt caaggacaga 27180ggacgatagc taccacgttg agttctagga aattaaccat tgttattgtt aaaggaagac 27240agcgtttctc agaggaagac tgttaaacag tgcagtggcc caggctaaca gccctcataa 27300gtgggagtat cagaatgagt ggacttaatt acttaaaacc aatacagggt ggaacttcat 27360ctgctataac agaaatcaac tcgtgcaagt tctaacatgc agggtacagt tctgagacca 27420agtctgactc acctgtcaaa gctcagctca actattacca cctttacacc acccttccaa 27480gctgtaggag tgcttgctgt tctccatgtc ttctgaagcc ctggatcact tgtagccagc 27540tcagcagact ctacccagac agggatcctt taaatgtacc atattgtcta ctgtgttaaa 27600aatgagagga actgactcag ggtgagagcg atggagtgtc cagatgttct cctttatttc 27660tccttattcc tggaaatgta atgagaatct tagaggtgaa ctgaaaagtt atgagttcaa 27720ccacttactc aattcgagat tcgctcctaa aatgtctctt ctgtgttatc acccccactt 27780tggtttgaat agtacttgtg acagggagct tatcacctca caagaaaatc cagtcattgc 27840ttgtagctct ctattaaaag ttttccatca tctggaactg aaatctggct ccctgtaact 27900tttagttatt ggaactactt gcccttcagc aacagtgtat gtatcctccc atggaagggc 27960ccttacatat ttgcagacac ccagcatata cttgcaatct tttcttcttc aggttcatta 28020ccctagtcct tttagttgtt cttcatttga cataatttca ttattcacta gtgaaccttg 28080ctgcccttcc ccttgataaa ccgaatttgt cagtgtcatt caagtataac tgacctcaca 28140gaacgtgata ccacaagcga tgtggtctga ttagcacaga gttcagtgaa tgaatcctac 28200actaggattg gatgaaattt acttagccat accacactaa cacttatgtg atttttatgt 28260ttactatgga tagactattt ctcctgtgtc cacttcttcc tcttacacag ttgttatttc 28320aaaactgaag tacagattct tacacttacc ctcaggagat tcatcatgtt agtattagtc 28380tctcttttca ggctttatga atgttaattc agctaactca tttttgagct atctgtctca 28440ttttgtgcca tctgcacagc ataagtttga tttctgttgc ttttattagt agttttacta 28500aatacataaa agtgaaatag tgaaacacag agtcttgtag catccactgt gggatcagtc 28560ttttagacaa gaatgatgca gttgctgagt caaatgaata aatgaataaa tcaaacaata 28620ctttgtcctc atttcccata ttgatctatc accatatcct gttaattata attctaaata 28680tttcttgatc tatccacttt tcccttactt cacctgctac tatcccagac caaacagcca 28740tcttctttca ctcaaacaat tgcagtagcc aactgattgg tcttcctgca tctgtcctgg 28800cttccctatc atccatttgc tacacagaaa ccatggtcat cttttcaaaa tgcaaatctg 28860atgatatcag tctcagctct aatttctttg gtggttcaca tataaagact gaaatcttta 28920actgaccaat aacacacgtg tgatctggcc cctgctcacc tcttcagcct tgtctttcac 28980ctgtctcttc attttggcca cagggacctc ctcgtacctt ctctcacgtg ccctcctgcc 29040tcagcgcctt tgcatatgct gttccctttg ccgagaactc ttcctgtcaa ctcccaagcc 29100cttcacctac ttagcaccta cctattcaat ctgttctgtt tgcctcttgg tatgttacaa 29160actgtctcca aacttagcag cttagaacaa tgaatccttt accctctctc acaatgtttg 29220gggtcaggaa tttgagcggg ccttggctga tttttctgtt cctcatgcca tcaattgata 29280tcacctgatg ttattaagct gatggatggg ctgatctgga gatgcactgt ccagtttggt 29340agccactggt tacctgaaat gcagccagtc ctaattgaga tgtgctataa ctataaaaca 29400cccacatgat tattgaagat ttggtgccac caaaaaattt aaaatattcg ttaataattt 29460gtattctgat tacatgttga gattataata tttcacatac atcagataac ataaaatgtc 29520attaaaatta atgtcaccta tttcttttta atttctttaa tgtgactact acaagttttc 29580aaattatatc tgtggcttgt aattgtggct tgtattgtat tctttttttc tgagatggag 29640tcttactctg ttgcccaggc tggagtgcag tggcgagatc tctgctcatc gcaagctctg 29700cctcccaggt tcaagtgatt ctcctgcctc agcctcctga gtagctgaaa ttacaggtgc 29760ccgccactat gcccagctaa tttttgtatt tttagtagag acggggtttc cccataatgg 29820ccaggctggt ctcaaactcc tgacctcagg taatctgccc acctcggcct cccaaagtgc 29880tgggattaca agcatgagcc accacacctg gcctgtttta tattcttact ggacagtgct 29940gatctagagc aggagtcaag cagttttttc tatgaaaggc cacatagaaa atgttttcag 30000ctttgcaggc catgcagtct ccatcatagc tgttcaactc ttccattgca ctgcaaaagc 30060agccatagat aataatttac aatagacata gcagtgttcc agtacaacta ttaataaaaa 30120taggtggtag ccagatttgg cctacaggct gtagtttgct gacccctgat ctagaagatc 30180caagatttta ttcatatgtc tggtggcttg gcagggatag gtggaaggct cagctgggac 30240cattgaccca aacagctata cagtcctctc cagcatgatg gtctcggggt agtgggacat 30300cttacgtggt ggctcagaac tccagataag gtactcccag agagacaggt agaagctgtg 30360aggcttctta tgaccaagct ctcgaagtcc cagaatatcc cttgtactgt attctatggt 30420caaacaggtc actcaggcta gcccagattc aaagagagga gatccaactc tacctcttca 30480tgggaggagg agtagccaag gatatgtgtt tctttttaat ctattatatc attcttcaga 30540tctcagttta ggctggtcct gttatgggct ctcaaagtac catgaacctc tcttttgtag 30600cacttgtcat agctagtttt acatttctct gtatgattac ttgatcacta tcttgctttt 30660ctactaaact gtaggcaacc acgtgaagag gaactgtttc tggttttgct cattatattc 30720ctagcaccaa acacaatgct tggttcaata aatatttgtg gaagaaacga atgaatgaat 30780gaaccaatag caaatgaatg aatgagtaat aactgtatca atattaatcc tacatttctc 30840catattgctg tcacgtatat cataagatac tctgtcagaa gccttgctaa aattcaaata 30900tatttgattc ccagtaacct tcttattttg tagttcagaa actttataaa gaaggaaata 30960agcctatctt actcttccca gtatctcaaa gagggtttct gccctgagct gctcaagagg 31020gtttctgccc tgagctgctg ttcattctgc aaacactgct cgaataccca ctgtgtgcca 31080ggtacagaga gttcttctct gctgtaatct ggacaggcac cagcttccca gcgtgggttt 31140aggcttcagg tgcacactac tgtgtaccgt ctaagccaca cctagaagag ctctggggaa 31200atatgactac ttgggcagaa aaggaaggaa ctaagaagag gtatctttgt gtctgaggtc 31260tgaaggagcg tgtgggctct tgttcaggca aagggcagga tgaggggagg tggggtggca 31320gcagccagta atggggtggg acagcggaat gcagaggatg aaacttcagg tcctggtgct 31380ctgagaagta acgctgtgca gcatgtcaca cccagaggca aaccaaggcc ccagggagct 31440gatgttgcac tggagctcta ctctcctctc agcgagctgg tgacgtgcca gtccagcagg 31500cctggcttat ccaaccacaa gtatgaatcg gcagaaggca atgagctggg ccctgagtgc 31560tgctgggctg aggccgacct aatccttcct ccacagagac tgtggtgtcc cctgctttgc 31620tcagggtaag aactcttgta tacctcacaa gaagccaagg actacctacc accttccaca 31680ctggccctgg agcctgcatt gtagttattt gtggacactt tttcttctct ttagtgccag 31740gtgggggacc aaggcctaca tgtctttaca acccctcaat ctctagaaca agtctgacac 31800tgagtagatg tagcaaatgt ttgcctgaaa gactacctca ataaataacc ttctgaggca 31860ccagcaaact tctcagcatt tttcctgata ctccggttac cactaacatt ctacacaaag 31920ttgtgaaata agtctttttc tttgttgctc tccaacatct actgtggacc cctcctctca 31980cttcctgttt catcctctct gcactcccct gtcccacccc attactggct gctgccattc 32040cacctccctc atcctgccct ttgcctgaat gagagcccac atgctccttc agacctcaga 32100tacaaagata ccccttctta gttccttcct tttctgccca agtagtcata tttccccaga 32160gctcttctag atatggctta gatggtccac agtagtgtgc acctgaagcc taaatccacg 32220ctgggaagct ggtgcctgtc caggttaaag tggagaagta ctctctgtac ctggcacaca 32280gtgggtattc gagcagtgtt tgcagaatga acagcagctc agggcagaaa ccctcttgat 32340gcaaagggat actttggggc cccttcttct cccaccccag tctgtctctc tgagagtcct 32400ctcgattcca ggagccacca tcacacctgg ccctaggctg tgctgctccc gtctgtctca 32460gaggctagat aacatcagag tcctttccac tggctcctgt ggcagagcaa aaactggttg 32520gcatttttaa acgtgctaca ccagtgtgtg aaagaaacac aggctgcatg ggtttaaatc 32580tcagctgtac catttactag ctgggcagcc tagggcaagt actgtgacct ctctgagact 32640ccattccttc atctgtaaca tggggacaaa taatctcacc ctgttgtgag cagtaataat 32700atgattaatc atttagccaa ctcttattca tgttctctga tgggccagac atacaaagta 32760agtgaaagtg gattacggca ggtgctcttc ttggtttctg gagtgaacct ccatttacat 32820ggaggctcct ctttttagat ttctgactag ttcacccacc ttattcatag accttattct 32880gtgcttagct gacagaaatc tcctctcaga gaatcccccc ggtaaattct taggttcttt 32940cctcttccat tccccttttt gctctctccc tccgaaggca agagtttcca ctttacaggc 33000ccactggaga aagttatggc ttctggttgt ggttggaggt tcattcctga gggagtgggg 33060acatttctac acttcttcac ggccaatgac attggagaaa ctggcttcct aacccagccc 33120acaccctcgc acacacacat cacacatcat ggctagaatg gagagaaatt cttcatatgg 33180ggcacttgta cttcatgaaa gaaaatcata tcaatcttga gtattttaac atcctattac 33240agcagggtca ctgataaact aagtgtccag agtgttttct aggatggtgt gtggtctcca 33300aattaacatt agtgaagctt actggaagga ttgttactcc tgggccaggc caggattttg 33360aggagagatg tgtttgctgt caccaaatcc ttgacagact ttggcagaag tgtgttaggc 33420ttactctgga tagcttcaga ggacaaaact agtattgacg gaaggaaggt aaggagaagc 33480agcttctaac ccaggggaag agagagtttc caaactgaga aatcaaaaat ggtactgatt 33540ccttgtcagg gtcagtgctt ctccccactg tgtgaattac aggggccatt tgtccaagat 33600tccttagagc aatactgatt tcatgtaatt atttgaatga aaggtgattt gttaaattta 33660tagtaaaata taatttgatt tgtgtccctg tttgtcatgc caccccagaa gaaaaattgt 33720ctttggttag gtcgaacata atggtttttt ggtttgcaaa ccatgagcga ttcccatatt 33780aggtgggagt tcagattcaa agggccctct tttttttttt tttttttttg tagtagccag 33840cctaatgagt aggaagttgt tctcactgtc attttatatt gaatttcttt tattttgagt 33900atgaccatct tttcaaatgt atgagatagt tatttccagt tccacatact atctgtacat 33960ttcttttgcc cgcttttagt ttgggtcttt ggcctttttc ttattgattt atagaagctc 34020ttttatacat agaaaattaa tactttgtga ctagttgcaa atattttcag ttgctgaaat 34080acacagtagg tgttccatgt aagagctgaa cagctggttc ctgattgctg tctccctccc 34140ttccagccaa tagatttcag agtttgggca ttacctattg agccaaagct gacaccacac 34200aagcgcagag tatgggaaca gagttctctg tctgattcct gtgagcttcc tcatactaaa 34260tcaccaacag caacctactt atcacagaat atgagaattg aacaagtgtt ggcaaggatg 34320tggagaaatt ggagctcttg ttccagttgt cgatgggaat gtaaagtgat gtcgctgcta 34380tggaaaatag tgtagcagtt cctcagaaaa ttaaaaatag aatgaccaca tgatctagca 34440attccccttc tgggtatata cccaaaagaa ctgaaagcag agtcttaaag agatattcat 34500acagccttgt tcataccagc attatgcaca atagccaaaa ggtggaagca actcaaatgt 34560ccatcaaaaa tgaatggata aacaaaatgt agtatgtaca tacagtggaa tatcatttag 34620tcttagaaag aaaggaaatt caaacacatg ctacaatgtg gatggccctt gaatacatta 34680tactaagtga aataagccag tcacaaaaag acaaatactg tatgagttta cttataccct 34740aagcagtcaa attcatggaa acagaaggtg gaatggtggt tggcaagagc tgagaggagg 34800agagaaagaa gagttattgt ttaataggta tagaggctta gttttgcaag atgaaagagt 34860tctgaagatg gatgtagtga tgactgtaca acaatgtgaa tgtatttcat accactgtac 34920actcaaaagg tgaagatggc aaattttatg tgtattatgc cacaactaat aaagatttct 34980aaaacttatg agatctaatt tcaccgtttc ctattgctaa agatcacaaa ttagaaaaca 35040cgttggcaaa aggtacatga aaataagcac tcttgtgttg atcagagcat aaacgtataa 35100tctcataaac taataaagat ttctaaataa caaagatttc taaaacttat gagatgtaat 35160ttcaccattt cctattgcta aagatcacaa attagaaaac atgttggcaa aaggtacatg 35220aaaataagca ctcttgtgtt gatcagagca taaacgtata atctcagggg agaacaattt 35280gcaactattc ttcaaccctt tggtcaaacg attctgcttc taggaatata gcttactccc 35340acctgtgtga tatggcatat aatcaaggtt ttccattgca acaaaagatt ggaaacaacg 35400ttaagtatcc atcactagtg gtctggaaat atatatatat tattgtcatc caatagaata 35460caatagacta atatgcaact tttagcatga ggatactcgt tacatgctga tacagaataa 35520tctccaaggt agtcatatgt gtgcaaaacc gtacatagta tgctaccatt tgtgcttaaa 35580aataaaaaga aaacagaata tgggtcaatg tttttgttta gttttgtcta aagtaacttt 35640aagtagaggc aagaaactgg taacatgtaa cagtgatcac ccctgttacc tctgtggaag 35700aaaactagac agctaaggga caaggctggg aggcagactt gctttccact atttatcacc 35760tttatctttc aaatttagta ccatctacat ttagtaccat gatctattca aaaatattta 35820ttaaaaaaag aaaaggtata gtctagaagg aaaaaaaaca taacagacac ttctagccca 35880atgtcctgca ctgggtgcta tgagagcaga ggaaagaaac acatatggct tctagacaac 35940accgtctggg gcatacattt ctgctattcg atcaagaata gttgtgcatc ttttcctgga 36000aagaattgat ttgtttttat caacagacct atgaatttag tggacagacc tgtgaattaa 36060ttcactggtt aggttttcct ttttacattg gctgttaaaa agctataagc caaatttatg 36120tccccctcag tgcaaattgg gcagatttct agggcaagca tttagcactg gccttgtcct 36180tggctctgta tcatattcct gtatttggtt tgcttttcca cctgtttctc atgttggtca 36240tctttcctgt gtatggccat accatcctga atgtgcctga tcgcatctaa tgttggtcac 36300ctctccttat tctttgcttc cttataagcc actaagcagc ctttttggtg ctagttaggg 36360taagtgcgtg ggtagtgaag gagggaggag ggagaggaag aaagaagata gaggttataa 36420agcaaagcat atcctttttc ttggcttcat catgtagatt aagtgaattg ctctcaaagc 36480gtggtcctta ggccggcagc attgtcatca ccttatgttg ttaaacataa aaattcatgg 36540gtttcatccc aacttactaa gccagacttt ctgtggttga ggcccaggaa actctccagg 36600tgatttttac tcacattcaa gtttgagaac cacaggaaaa caaaaggaag gcagatttct 36660aagcgtaaat gcaatactaa ccgattgccc ccatcatgcc tgttatgttg gtcaagataa 36720ataatactag ctactgcaat aatcaatccc tcaaatttta ttttttgcca atatcacaat 36780ccattgtaga tcagttgtgg gagaggtgta aagagagctg ctttattagt ttattaagca 36840aaccagatct cttccattgt gagactttgc gattttctag gcccttggac atttcctctg 36900gatcccctgc tgctaagaag gcaggagagg gaggaaagag aagagacttt agcagccaga 36960tctggaagaa acatcttttc tgcccacaat tccattggct agaagccagt ctcatggcct 37020gtataactgc aggggaggct gggaaatgtg acctatcgat ggagctaaga gcaaaaggaa 37080atggctttga tgaagccctg gcattgtctc tgcacacccg agaacccaag tgaatcccaa 37140actccacgtc caggtcatgt tttggtgaac atcggttttc agtttccttt tctaatcaag 37200ttttaccttt ttttttctcg actctagcac tatgggactg agtaacattc tctttgtgat 37260ggccttcctg ctctctggta agaacctttc agctttgtta agtcctggaa tcctactgtc 37320tcctgatgag tctgaccaca gcaagcccag gcctgagact tggtgggttt tactcacttt 37380ctactgagca ttgtacaaga ccacatgcaa aaaagacttt cctggagaag aaggaagtgt 37440tatgattgag agcagctgat ggcaggcagc tgggatggag ctctcccccc cgtgtgcttc 37500ttcctcctct gcagtctcac atcagtgagc ctagatgctc agagtagggt agcctggccc 37560atcccatggg gatgggggaa ggctgctgca ctgaggcccc tgagacttga ctcttttgtt 37620ccacacatat tctcttctgg tcttctctga ccctgtttct gtctttctca ggctcctagg 37680aaacaactga cagaattcca aaagtctccc ttcattcgga gcactggctt tcacgtccct 37740gacttcccta ccctctctca ctcccttccc tacagcccat gcacatacct catggttgcc 37800acggcttcct gacaactatg gatgttcagc taattgtgtc agctgattta tagtggagcc 37860aatgaagctg aagcttcaga gccctccatt tgcacaaccc tttctaaatc cccctcaaga 37920ccctgtgaag ggccccctag cagtgtggtc acctgtctta tgctttggta aaatttgaat 37980aagtaagata ttgtaaccac aataagttat gaccactgtc tccttcctct gcaacttttc 38040cctccatgcc attctcctgt ctggtggtgt tagcagtcag gggcattttg tatttgaatt 38100ctacattctt tttcttaact atccaccacc tcccctcaaa attttaacag catccagcct 38160cacaaaactc agatcttccc tgtttacagt tccactttga gtttcagttt cttcatctat 38220aaacaggagt tggctgcggt ccctgccatg tatcctgtga ctcagtgtct cgtagttact 38280cctggcccac cccttcctgc tgctccttgt ctccacctgc aggcctgaga gggaagccac 38340cccactaaga cagggaggtg aactgagcct gaagtttggc tacagcaccc acaggccacc 38400agccatgagt tcacctcctc cagatggcca cacaccaggc ccttggccac tgtccccatg 38460tctgctgtgg atgatgagga gtcagggaac tacaaagaga tggtccctca gatccatgct 38520ggctgggata agccttttca gatttctgtt tttctgctta gcaccttgag cttgtggagt 38580ccttgagtgc aaggtctgta gatgtgccag ctgatcactg acttaggtaa caacagcagc 38640ttccaacccc cagggcccat gacctgctac cttagctcct ggggatgtgg gaggtatgtg 38700tgtgtcagag agcaaggcaa gaagactcta gagaacatta tccagtaaga ttcccttctc 38760atcccacttc ttatttattt attttattta ttttattttt tgagacagca tctttctctg 38820tcacccaggc tggagtacag tggcacagtc acagctcact gtggcctcga ttacctgggc 38880tcaagcaatt ctcccacctc agcctcccca agtgctagaa ttatatgcat gagccatcgc 38940acatgactta ttttatttat ttgataaatg catatataca cacagtcatg aatcgtttaa 39000caacaggggt acgttctgag aaacacatta ttaggcgatt ttgtcattgt ataatcatca 39060tagggtgtcc ttacacaaaa ctagatagca tagcctgctc catacttagg ctacctggca 39120cagcctattg ctcctaggct acaagcctgc acagcatgtt actgtgctga atactgtagg 39180tgttgtaaca caatggtatg tatttttgta tctgaacata tctaagcata gaaaagatac 39240agtaaaaata tggtgttata atcttatggg accaccattg tatatgactg aaatgtggct 39300gtgcaataca tgacagtata tgcatatata tatatatccc ttactttgtg cctggtactg 39360ttctaagtac ctcataaata ttaactcatt tgagcctcac aataactctc tgctttaggt 39420cttgttgtta tttcccattt taagatgtgg acactaaagc ccagagagat gaagtaattt 39480acccaagatc gacagagcta ctaagtggca gagcttggat tcacacccag caatgtagat 39540ttagcattcg ttcacttgac tcttctccta actcttgtgg taaaccatga ataagtggta 39600agacttcttc catggggcct gaacagcttt ggtggataat atagcttctg cctcatccgt 39660gttcatccag tgcctcctcc ccatcacctg cagctgacac ctcagttgac ccaagagctt 39720gggcccaagc ccttctcatc aaagtgacca gcccagctct caagatctgg gagagaagga 39780agaaaaatgc cctggaaaca catttccaga aaacactaaa ctggaacacc atttcccacc 39840aaattttctg actccgcaca ctgaaagtga gaaagtaaag ccgagacact ctatgaaaac 39900tgagttcagg tgtcactttt gcccttgatt tgccattgac acttcttaga agtttcttag 39960ctcctgagaa aagagttacc aatattgaaa gcaacaacct caaatggtaa ccgtttaagt 40020tttatggtgg tgagagaata agtgactata tttttggcag tacaatttta aagtggaata 40080gaaagcccat gacatcagat cagaaaataa cattgccagt aattcacaca cgatgaaaag 40140caacaaaaaa tcagattcta tttgaattct ttcttctcag ggcacacctc tgcttactgg 40200gctggtgaac agtgacctag ccacagggcc ggcttccaaa gggagaaagg agatgcaatt 40260ggcccacata atccaccctc aaaatgtaga gctgaataat tcatttcatg gcatagaaat 40320agcaatacag tgaagcaatt ctgtttaact tttccctccc tatattttgt gtcctctgtc 40380atggaaattt gacacagtag tatttgctgc ccctgctctt gaggataaaa ttggatggga 40440gtttaagact gaaacgggca cctgtggcct tgcagaatta ggttacagtt tgtgccttgt 40500atttacaaag cgaaaggaat tcctagtgcc acctgcagag gcacttctaa ctttcaagct 40560ctgtttgcca ctgtcctggc acctccatca cacttttagg ctggagccag agaggttttt 40620gaaaaatcag tagctcccac atcaggagga agtatctttc cagtttgagt tttggtagct 40680gctctctttt tgtctgaggg ttctctgggt cctagggctt tctcatttct cttgaacaac 40740acctctagtt aatttcatgt acctggagtg gtagttggaa tatttcttca ctttaagatt 40800tttttttttt ttttttgaga tggagtctca ctctgttgcc caggctaaag tgcaatggca 40860tgatcttggc tcacggcaac ccccgcctcc caggttcaag tgattctctt gcctcagcct 40920cccaagtagc tgggattaca cctaccacca caaaatacaa aaatacacaa ataatttttg 40980tatttttggt agagacgggg tttcaccatg ttggccatgc tagtctcgaa ctcctgacct 41040caggtgatct gcccgcctcg acctcccaaa gtgctgggat tacagacagg catgagccac 41100tgcgcccggc ccaccttaag atttatgtaa gattggctca aaagctcatt cctgtggaaa 41160ggtccactgt tttcctccca agatttttgc agatatctgc gtgggtggtt acttttgact 41220cccatttcct gctgttgttg atagccctca ttaaaaccat cacctggagg tgaatagaca 41280gtcgagacct atcattccca aagaattgtc atggagccta atagttctat tggattcacc 41340cctttatgtt aagccaccat ttcagtgttt ttcaaaatag atatatgtta tctagtaggg 41400agtatcttac ccccaaatta gttgattgtt tcaggagggc ttttagtggg ttccagagaa 41460aatgagcaat cagacaagtt gatttagtgg aagacagtca ctgaatagga tgtgtatagg 41520gttgtttggg agcaagagtg aaattggtat ggaacagaga ggctcccaag gcaagcagac 41580attttttttg gaagaagcaa gtgtttgaga gactgtggct tatttttcct ttgtgagagg 41640ggagttttaa taccatttcc aaaatatgta acctggtatt ttgtccccag aagtactgtt 41700gagatttatg gaagcaaaaa actctgtcac ccaggctaga ggagtgcagt ggtgctatca 41760aagcttactg cagcctctaa ttcccaggct caagagatgt ttctgcctca gccacctgaa 41820tagctggcac tataagtaca tgccaccatg cctggctagt tttttttgtt gttgttttgt 41880tttgctttag agacggggtc tcgctttgtg cccaggctgg tcttgaactc cttttaagtg 41940attatctctt ctcagcttct taaagtcctg ggattatagg catggcctat ctatttttat 42000gttttataat ttcttgtact ttttgatgtt acttcaaata tctttttaag tatcctaaat 42060atacttattt aaattttttt tgagtaaatt tatctataaa ttattgattt tatgtcgata 42120gacattgttc tctatcatta ataatgttaa aaataaataa aaaaacaaaa acaagtaaat 42180caattaatgc ttaccacagg ccagtatttg atccaacact aactcaaata ttcatttctt 42240taatcctcac aacaaaccta tgaggtaggt accattattg ttcctgcttt ttgcaagagg 42300aaactgagac acagggaagt taagtaattt gcctatggta acacaggcag tgagtagttg 42360agctgagatt gaactcacgc tgtccagaat ccatgctatt agttataata gtgtactgcc 42420ctatagcttt ctgtttcaca gctacatggc attactttgt atggatgtat cattatttgt 42480taaaccattt aacttatttc cagtgtattg ttcttataaa caatgaatac ctgtgtacct 42540ctaattttgt gcacatgtat ctttttgtag aatgaattct taagaaattg agttgctaag 42600tcaatgctta agcccataat taattttctt acatattacc aactgtcctc caaaaaggtt 42660gtaccaattt agaattttac cagcagtaaa ttcagcagtt aggacccatt ttcctaacac 42720tctcgcggac actgggtatt accagtattt tttttaatac gtgccaatca aatgggcaaa 42780aagaatggtt tctcactgag gtttaaattg catttcccta gttattcttg agatttttcc 42840tttcctttct tcaacaatta cttattgagt gcttcatatt tgtaagggac aattgcaggt 42900actggaaatg tcacagtgag gaaaagtgac aaagcccctg ctgtcatgga gcttattcta 42960atgggagatg tcaggtgctc agctgagctg ggagagagag agctgagttg tcaggtgtca 43020gaggagccaa ttatagcagc aaaacaaaaa taaaatagtt cagcttttaa tctcttacta 43080cgacggtata atcaagaggc taaaatggga ggaagggcag actctgcctg ttccatttcc 43140ccacatagag tgagtatacc agtcgagggt caggtaatca gtgcagactt agggggtcgc 43200cttaccattg aagaagcccc aaatgaaagg ctctagcagt tttatggacc tgggggtgga 43260ggaatccaag ggtggggaga attcatgagg aaaatgaggt gagagggcta ggagtggaaa 43320agtacaaagt actgagttag cgtggggaat agtgtcttta gggctaggag tggaaaaaat 43380actaggtact gagtcagagt ggaaaacagt gtcttcaagg cagggagtgg aaaagtgcta 43440ggtactgagt ccgagtggag aaaagtgtct tctctatgat gaggaggctt cagcagaggt 43500gcctgaagac ctcaccccag agcctcagat aaagagacct aagaatgagg gtgcctgggc 43560taagattgca agtatgtgaa aaagcatgac tggcgggagg ctgagatctt gattgcagcc 43620cccttcagag actgccatgc actgactgtg caccaagtct gctgtagaaa gggcaacttc 43680ctcagcaagg cttgtcagat taagcctctt taattgcctg tggtcaggtc tgaaaaatca 43740cacatagatt tttaatcaga acccagacat ctcaggagag acagacaata accaaacata 43800ccgtgtcatg tcatgtcatg ataagtacca caataaatat aagtcagcat gagggacaga 43860atgcccagga tgctatcttc aatagaatgg ttagagaaat ctccctggga ggtagcattt 43920aatgaaagac ctacatgaag tgaaggagaa gctatgagac tgtctggagg aagaaccttc 43980tggacagagg gaacaacatg agaagaggac ttgagacaga gtgtgtgatc ttttggagga 44040atgtcaaggg aggcagtgtg gctggggaga gtaagcaggg gaaagaggcc tgataggtac 44100tggggaccca attacatgag gtcttgtaag gccaggggaa ggactttgga tgtagttctc 44160agtgtgaggg gaagggatct ggatatattt ttcagtttgg tggaaggcat cagaggcttc 44220tgaacaggag gattatgtga ttggagctgt atttttaagg gatcattttg gcttgagaaa 44280ctagacccgg ggacaaggac ggagcaggca gatgagttag gagacaatta cattagtctc 44340ctctaccctt ttcttaacat attggagttc agctctggct gtagtagttc tagatctcct 44400cagacacact tgtgtagagc ctctgttggg tattttgggt acacaaatga ttcatcttgg 44460ttatacagat gatttagatg attgtagaca gaagagggtt gtctggtcat tcccagacag 44520gggagcattc cttgagatag agtagaggaa ggctgaaggg gaggaagaca gtacctgttg 44580ctatctagat agagacatcc agcaggaagt tgaatacagg tatctgaaac tctagtgaaa 44640gttataggct ggcaataagc acctgggagt tattagcttt tacttgacag ttgaatccgt 44700ggggctagag gagaaaaacc aggaaagtat ggagaataag aagaccaaga acatgcactc 44760aaggttacca aaattaaaga gtgatttgag aaaattaaca aggaaatcag agattgggaa 44820agaatagagc atttcaatga ggagagatgc caacacttgc atttgacaca gcggtcaaat 44880gagttgagat ctgaaaagag ctcaagcctt ggccatggtg tgaagtcacc aacaaccttt 44940gtcagggagt ttcagtagag aggtgggggt gggaggctgg gaataaaggc agcaattgct 45000gcttactctt tcagggagtt tgactccaag ggaaagagaa actaaaagca gtagcacaag 45060gtttgtgttt gaagtaatgg aggtgaacca ggtgaatagc ctggaggccg agtgaagtga 45120gacaggacac tgcagatttg gaatgtcacc agtccgcaca actgaataat ttcctccaga 45180actgctcaat tgcccagttg taagaacaga tatgtagacc aaaagtagag tgtccccagg 45240gtaaatttta tagagacaaa ggggtgtgtt tattgaagtt gtggaaagga ataattacaa 45300agacatacta ttgttgcatt gtccaatata ataaccacta gccatatgtg actacttaaa 45360tttcaattaa ttaaaattaa ataagattaa aaattcatct tctcagtcat actagctatg 45420tatcaattgc tcaatagcca caggggctgg tggctatcat attgttcagc acagagacag 45480agcatttcca ttatcactaa gagttcttgt ggaaaacact gcactacagg gtctggataa 45540agctgaggtc ttgattaagt tgaacaacag ttgtagaagg agtaagcaag agcaaaacct 45600ggatgaatag gaggttgtgg acggagatta gtatattgag attaagattc tagggactga 45660gctgctccag gtgaaaagtt tcagggttat gtcataagaa ggtggggggc agctgctgaa 45720atagtctgcg ggtgtagacc tgtggagttg acaagatcaa agaaatttga ggcaaggttg 45780ttagactcat tcatgaagaa gtcacccaaa ttgttagcaa gaccttgcat ctaatgccaa 45840aatcctcatt tagcaaggtg gtagtgactt agtagctaca agcaatgaga aagtcagaca 45900cctcaaaagg ggaaggtgtt gctcaaagtc cccacaaagt gtgataaaac aaacagtagc 45960tggggctgga gcaagtggct tcctttgggt gaagccagat ttcactgaaa taataacctc 46020agggaaacag tcaatgaagg ggttaaagat gtgggagagt ttccttgtag taagtaatgg 46080aatgaggctt tcaaagggcc aagtaaaact ttggaggaag tttagtaaaa gaaggaattt 46140tttttagtac agataagcat aggaacataa agaagagata attcttaaac atataagata 46200tgcatttggg gatagcagcc agggaacact gaagtcccag tggggtcaga gacttcataa 46260ggctagcaaa ttacagtttt tgagtggcat tccaacagta gagtgtattg ctcaggaagt 46320ccttaattat cctttgaaac aaattccttc agctgattac gaaggcatct agctggattc 46380ttgagcgact tgttcctgac atcatagcaa cccattgtaa ctagacttcg accattcctc 46440ttacccaagt gctggggaag ggagagattc tcaatgctta cccacctatg gaatcccagt 46500aagtccagtt gctaggtggc ttgaggtctg gggtcataaa atggaaggcc tgaagtcatt 46560tggtgatcac agaccttgag ccaaactttc cccatttagt cagagaaagg attagcagca 46620tcccccatgc ctggctctgt gtgagatcat ggaagccagt ggttggtgag gtgctatgga 46680gtataaattg caaaatactt tcagttccac tcagaatgga tttcaaagtg atttccaccc 46740catggggagg agagggagtc tgaggaggga tggatggaaa aaaaattttc atgtcatttt 46800ctgtgatcca ctctggagac agaggcagag attctctaca acagctgctc aaactatagc 46860tcttgttaaa atggaggttc tgaatcagta agtcttgggt ggggccagag attccgtgtt 46920tcagaccagc ccacatgtga cgtgaatctc attggtccat acatcacact ttcagttgct 46980aggtgaagaa gggagcactc gatgagtgga agagaaagcc gttgtaatct ttgggagaag 47040gggcctgggt cagcggagtt agactggtct gtgagtggac agaatggatg ggaaggaaag 47100aagatactgt gaggctctac agaaaaaaaa aaaaaaaaaa atatatatat atatatatat 47160atatatatgt aaatcaagaa gacagaagca gctaaagacg aagtcatttc caggtccaga 47220aggcacaact gacagctgag taataacata acattgactg ttaattggca gaatttttaa 47280ctgtgtgttt ggtttctcca tcaggtcatc tgtcctatat tacatgacaa tttagactaa 47340aaccagtatt tcctcagaga caatgctaga agcttttaca gtagggggca ctcttgcatt 47400acattaagag ctcagcaaag aagatgcaga agcctcaggt ttgccttgta aggtgattca 47460taaacacact aaatcttcct taggtctccc tttcactgtc agggtacgca tatagatttt 47520ccttcctccc tccaataccg gtacgcatcc tctacaggtg gtgcatttta tacctcaagt 47580acttcacagg gtcctagtga gtgtagtgaa ataggcagtg attcatattt gtgcaaactc 47640ccactgatgc ctgctgtctg cttccctaag agttcaagac caccaccaac cccttgatta 47700tgtgttctca ctgggccact ctgtacacag tttagtttga caagtgcatg tcactgttat 47760ctgtccttct attccctctt tcaagagaaa ccacatcaat ttaattactc ccccacttag 47820aactcttcaa atgaagctcc tctcatctct ctcatcaacc catctcctcc ctttcctcct 47880caatgtcaac atgccttcac ataaatcctg aatgatgaaa ttttatttag aacttacact 47940aacttcctct ccaaggtggc atctaacttc atattaagta agaaacagcc ttcccactct 48000ccacccccgc acttctcacc caccactgct tacttttttt tttttttttt tttttttttt 48060gccaagtctc aagtaattct gtaacctaga aaaggtccta cacaaacccc gtgatcattc 48120acatttaagt agttgggtgg cccacatcct tcccacaaac cccaaagtgt cctcaaggac 48180taaagccttt ctctcaaccc ttccagcatg atgtctatgg ttgtaaaatt gtccagggtc 48240agtgcatact gggagcagca agtttgtggt gcctggggtt tccccaatac tcccaaagca 48300catcctcacc tgcccatcta tgattcattt tcagcatttc actcatgtgc cttaaatggt 48360cattgaccac cacaatccga aaacagccat caaatttgcc cagttctctt tctgatctct 48420gaaagagctt agagaggtca ctgaaaataa aggccttggt tcactatcga agtcatttct 48480aaagcatttg acatccttgg aagtgctggc catgggagca gcagtcatag gggaagttct 48540gtaaagggag ctatttgaat ttcaaagatg ttactcaacg tgattcccca actaatgaag 48600tataataaag gggggctata atttattacc attatcagca atcttttcac catagcagac 48660caaggaatat gtggatggga ggggagggga aagcttttgg tgatggtgta gaagttatgg 48720aacctgtaac agctacagtg atgaaaacta aaattaaggt tataggaagg taactggtgg 48780gtgaatgggt tgtctaactc tactggtttt tccctgtctt gcaatttaaa ttcacagaac 48840cacagtacta gaaagaccct tggaacattt agtcaaccac ttcattaatc agatgaggaa 48900actgaggctc ataaagattg cagtttgtac aaggccacac atttagtcag cggtgaagca 48960aggacaaagg tcctaatctc cagatgccaa gcagatgtgc acagttccag agcttaatat 49020cttattcttc agcatgatta ctgataagat agtatctggg tattgtataa agagaaatgg 49080aggttttttc ccctttcctc ttgtttctcc ctccctaatc cttaaccttc ttttttaggt 49140gctgctcctc tgaagattca agcttatttc aatgagactg cagacctgcc atgccaattt 49200gcaaactctc aaaaccaaag cctgagtgag ctagtagtat tttggcagga ccaggaaaac 49260ttggttctga atgaggtata cttaggcaaa gagaaatttg acagtgttca ttccaagtat 49320atgggccgca caagttttga ttcggacagt tggaccctga gacttcacaa tcttcagatc 49380aaggacaagg gcttgtatca atgtatcatc catcacaaaa agcccacagg aatgattcgc 49440atccaccaga tgaattctga actgtcagtg cttggtatgt ggtcaatggt gtgtgttcag 49500attcttagcc ttctcagatg agactgcaaa tgagttagaa aaacactgga gggggacttg 49560aggggcccag gggaaaaggg gggtctatag agagaaggca gaggacagcc acttctggga 49620agtgcatttg aagggagtgt agagtctggg agtagggaac tgaaagtctt ttgtactttt 49680tatagtctgc ttctgaagga tcagtaaaaa tctgctttgg ggaaaaaata gagctaattg 49740aacaaagata atatggctaa ttacctatag taaaaaccat ggataatttg gccatcacaa 49800agtttatata accataaagg cctcagatgt cttacattca ttttttcctt gggtccaaga 49860tttttcacct actaaatctt tgcctggagc tcctagcaaa gcggacagct gacacatttg 49920ggttttccct tcagcctcct ctaggttgct tatgagttgt ttgctgccac aaccatgagc 49980ctggtagaca gaagggaaaa aaacccaaca aacataaccc acaaacttac aaaccagctc 50040ctctgcttca cgagaccttg gaaggcctaa atgccactac agattttttt aaaactatca 50100cacagtaaaa ttattttttt ttgttttgat atactgttct actgattgta tagatcttgt 50160atagatttag gtaaccgcca caggacatag agcatttcta tcaccctaaa aatttccctc 50220aggctgtccc ttcatagagt cataccctgt ctgcactcat aacccttgtt gggcatccta 50280tagttttgtc tttttgacag tgtcacataa gtgaagccac acagtatgta accttttaag 50340cctggcttct ttcgtttagc gcgccttcga gattcaccca agttgttgca catatcgagc 50400ttgtcccttt ttattgctga gtagcatttt attgtttatc cattcaactc agtaaaagac 50460attgggttgt ttctggtttg gggctcttat gaataaggct gctgtaaacg ttcatgtaca 50520ggtttttgtg tgaacataag ttctcagttc tctagaggaa atacccaggt gtggtattac 50580tggatccagg ttaatttttg atgaaacttg aaaaggcaga tcaacaccta ttctaaaacc 50640atagagtaaa acagaagcaa aagtaaaaat agaatggaga gctgctccct ttgaaccctg 50700tgtgatttaa actaggctgc agggctttag gaatagttaa ccaagtgcta aatccgtgtt 50760ttcaaaatgt ggtcaggtac cattggaaat gttttaggtg ggacacagat aagcattttg 50820aaaagccatg ttgtatttgt tttaatgtat attagaaaaa ctctaactta cgcaacatgt 50880gatttcacag atcttgttaa tgaagctaaa cacggtctgg caattcacct tctacaggcc 50940acatagactc caagaagact gctcaaatag tacactgata tagcaaaact tataaagatg 51000acatgcaaat gacagacctt ttagtaagaa tacactaaat tataaattag tttgtagaac 51060ctgcaaacta cctagtaact ataaaagaac aagggatttt ttctgacaga aggcacatga 51120cacaggtcta gggactccat gccagtgatc ctgaacagcc agaaaagtga gaatggcaaa 51180ggcaagagaa acactgtgtt tattaagatc atgtattttt ccctaaaata gctggatttg 51240gccttcttct tagagtatgt tatgaagaca ctttgatgct catgccaaaa atcagtgttc 51300tgaatttcga attccaaaat atccacccac tcacttacca caatcctgct tgggtttctg 51360aaagatatga cgcagggcat ctcagcacca tgaactctgt cagttcctgg tgagactcca 51420gctcaattcc ttcctgctct cttagtctgg ggagctggaa tgtgccccat gggacacctg 51480ggccctagag tcagaccact tctccttcca aagactctac tccctggaaa cagtggcttc 51540attgtaaatc tttggtgact caattacagc cctcctgtca cttagagagc acccctttga 51600tttggataag caggaagtaa gcatggctgc aaactctatt gttgaaaaat aaacatgaag 51660tcattatgtg gcactcacct tgggctgagg gtcacatttt agacaccctg aggctcccag 51720gtgtgcccca atgagcccca gatcaagtac ccagttattt gctattccct cctagataca 51780tctaaactta gattgatttt tttttatctc tcttctgctt tcagctaact tcagtcaacc 51840tgaaatagta ccaatttcta atataacaga aaatgtgtac ataaatttga cctgctcatc 51900tatacacggt tacccagaac ctaagaagat gagtgttttg ctaagaacca agaattcaac 51960tatcgagtat gatggtgtta tgcagaaatc tcaagataat gtcacagaac tgtacgacgt 52020ttccatcagc ttgtctgttt cattccctga tgttacgagc aatatgacca tcttctgtat 52080tctggaaact gacaagacgc ggcttttatc ttcacctttc tctataggta aagctgtttt 52140ccaagactat ttctttcagc aggtattata cacaaatgct taaggcagat catccaatgt 52200ccccgacttg ctaggaaacc tccaactggg ccattttatg acgctgttag gaaggaccca 52260gatggaggtc tcctgcttct cctgagtgat gcagggtcca ggaggctacg agcctatgtt 52320gcacttgaag aaatatgctt ttagccctga aactgactca gtctcttggt ttacctttgg 52380atggaggatt ctgaagtttt gatttaaaaa tacaggattc ctccaggcta gaattctttc 52440tttgattaca acacatacat gcgcttgcac acacacacac acacacacac acacacacca 52500tgcatacatg cagacataca aatgatattt attgtgagta tagaaccatt tgggacatta 52560ttggtcacag gagtgaaaac aaaaagatat gacaccccct ctgcccttga ggaccttcca 52620atagaatcag aaccctgtaa tgtgcacaca tgaaaaactg gatttttaaa aggttgaatt 52680ggaatctaaa ttttattcca tggaaatatc tgactaaatt taaaataaaa gtgactggta 52740atgagattta tgggcattca gaggtaggca agatccctga gggtcaggga atggttccta 52800aaggaagggg taccttgtaa catgtaaaat aaattattgg ggttaataaa tgtggtgagg 52860aggggagggc attctggatg acaggttccc aaaactgtgg tgacttccgt agctgaaaaa 52920atttgagaca gtatctgggc taagcaggtg agaggaccac agtggatcag ctgtatctga 52980cgtaagtgca ggaggtatgt caaagaaagc cttggaggca gaaatgcttg tgtgttcaca 53040agtattcttc agggacaagt tcagtggagg aaaggattga aactaagcag tagccactaa 53100taggagcctg acattttaaa gtcctggctt tacccaggag ggcatgtgtc tatatttgac 53160tcctctttta agaagctgta actgcaagat tccctcctgg aataaaggtg gtctgcatct 53220accctgtccc atcactgcct gtgctgacct tgacacccac atctgccttc ttcttacctt 53280gaccccttct ccagcggtga tttcttggct tgccccctcc agtgacatcc atccaactcc 53340ttgctccata ccctggcttt gtcacctcct ttctcccagt gtcttgttgt tcagatataa 53400cttggtctgt gaacagccca cggggccagt ccccatgaac caactttaca actgggccaa 53460tctcatctcc tgctactgac ttcttcctat tcagacactt cagcctctga gaatccagta 53520aatggtggag ccaactcgtc ctgtcccagt tgcttctcct gtatcctctc ttggccagat 53580agaagcctct ccaagctatg cctgaagttc agtacctcct tcaatgtgta attagtttga 53640ttggtggcca caagatggcc atatatgaca tgccccaggg ccctctgtta cggctcccat 53700agtctacaaa ttaacagggg cttgccacca ctataacctc atcatggctc accttcctgc 53760tgcttctcaa ctactgttct gccaaacttc aacaggtacc cccatcttca gaaatgtttc 53820agctctagct gcctcaggaa gatggggctt gcctctctgg gtttcccatt ctatcgcttg 53880atcagagata ggttagaccc tgagtcaagg ggcctttttt gcatgttaaa aggtagcagc 53940ctccacgtta gtaagtataa cccctaaccc cctttactgg gagtgccaaa ctggctcaag 54000tggaatagac tgggacagac tcaaaaggga ttaaatatgg cctgcaatgc caacaacttc 54060ttaacatccc agaaacaggg catgtgtcta caaattatag ctaagctaat agatcagctg 54120gtcctaattt tcctgaaatt tgggattagc taccagaact gttcccaaaa atgtctttaa 54180agtgggcgac tccgttctaa gttttcccca caaagcctgt tttccaactc cccagaaact 54240taggagttct catgtaagga agtagttcct gaaggcgtga aggttcctca aggcatgaag 54300aaacatcaaa ggtttttcag tagatgagat atgctgaaag ccatgcagag gaaacctgct 54360gtgacctcag taggaaaaaa ctaaacaaac aagcaaatga aaactagagg taggggcctg 54420tggaagctgt tccatttgtc caagtgagag gtgtctggag attatagtgg acagaagaat 54480catcacgaga ggaacttcag ggcctgggaa ctgactgcag aggggggcag gatagcaggc 54540acggcacaaa tgactgcacg tgcagagcct cagcacagac acctcaccca gattccagaa 54600tcacgggcca ggctgaccct cttcttcctg atcatggtcg gtgttatccc cacctccatg 54660aaggcatggc agctcagtcc aggcatttgg ccagaggcat gggctcgatt cttaggtcgc 54720tgctgaggcc ctgagcctgg gactttctat ggcctcctat tgtggatttc aggcttctct 54780ggccttagag ccctggggag aggctggcag gtaaataaag agaagagcag ctagcagaaa 54840ccttttgtaa atgactctcc tggctgattg aaaatttgtg gtcatttgta gagcttgagg 54900accctcagcc tcccccagac cacattcctt ggattacagc tgtacttcca acagttatta 54960tatgtgtgat ggttttctgt ctaattctat ggaaatggaa gaagaagaag cggcctcgca 55020actcttataa atgtggtgag tgagtccttg tcctccccac agactgtcac tttgcaccta 55080cttcccaatc ggctggctgc cttccggagc ttgttggctg agcctagact ggcaaaaagt 55140caggaagttg ttgggaaaaa aggttttccc ttggagtttt gagcctatac agactggcag 55200tagcagataa tgctgctctt ggacttcaaa gaaaggcgac atttctaacc tctggtttac 55260aaatgtactt ctggtttcca gggaaaactg attattactt gctttatcta cctcacttca 55320tgaggttact gtgacatata cataaagtaa aatggtgaaa ccactcctaa atgttaaaga 55380ttgtggacct ggtggtgttt aagcagggat atttgctaaa tgaccacaag aatcagcttc 55440tcgtctctaa aaaaatctag gtttcttatg aaataagtta gatgaattat tgcccattga 55500cttataacaa acaatattaa ctttaactaa tttctaagta atacatatcc attatcatat 55560ataccaaaaa taaaataatc tataactcca ctaataagaa aaaatgatta cacaaatatt 55620tttggtgcct atctttaaga tttttctgtg tatcaatcta tgttgttttc cataattagg 55680attatcataa gggttatttt tcacaatttg gataatatat gtactgtgtt ctaattttgt 55740tatactaaat gtagcaagac aattttcaat gtcataaata tcattctaca gcatcatttt 55800taatggctgc aagatattcc cttttgtgga tacaccataa tttatttatt taaccaacct 55860cattttttgg acacttgagt tagtccaata gttttgttat tataaacacc ctccccactg 55920acttctgtta taaaaatgtt tcatggggac aaagtggtcc ctaactttat aataatgcca 55980tgcctttttg tagtttggtc tggttctaag ctaagattgg actttatctc agtaattgcc 56040tccagtagta attagtttga ttggtgctaa taattaaggt aaccttctaa ctcacttatg 56100gtagaaagca caagatgagt attgcctctg gccagcatct tgtttttcag tatactgatt 56160ttaaaatcta actagaaaat agatggatga cattagcagt cattcaatgc atcctgctgt 56220actttaaaaa taagaaattg gggagcaacg atcgaattta aataaattaa cacaaagcat 56280gtggcagagc cattcaaact gccaatgtat ggagtgtgct gcgagatttc tatgatataa 56340aagtataaaa ttcctagcac agatgtaaag acatatcatg cttgtccagg ctttgacttt 56400tcaaggtgag agttttgagc ttcactttct ttcaacctca ttgccattta aaattagtca 56460aatatgaaga agtgacttac atcttgggaa taagctgttt gctagatttt tcttcacatt 56520agaatgatca gcttacaaat gaaacaaaga agggttggag aaaaagatta aggatgtttc 56580ttcctccatg aggcaatcag aaaaaaatca ggagactaga taggggagat aaagaggata 56640tgtgtgttca catgagagaa gttagaaggt ggttaaataa gctctgtagg tacagatgag 56700atggtcagat tgggctgagt ggcacataca tgacccctaa gaatgtaatg aagaatattg 56760gtaagaaaaa gttatttatt cagacagtca tccatgccac tgagtttgat caaagagaga 56820agccttgcta tcactgtagg gagggaggtg caacaggtat aactatgcca ttatagatat 56880gatatatttg taaatttgga ttctgtaact tcagcaatat ctgccattgc tttgtgggta 56940ctcctggcat tggctatgtg ataggtaaaa taatgccccc cacaagacgt ccacctccta 57000tactccagaa cctgtaatat gttatcttac atggcaaaag gaacttcaca taggtgatta 57060aggcaccaag cttgagatgg tgagattaac ctggattatc caggtgggcc caatgtaatc 57120acatgagtca gagaaccttt cctagctggg atggagaaat gaactggaag aaggagagat 57180ctgaaacttg agaagctcaa cccagcattt ctagctttga agatggaagg aggaagccat 57240gagccaagga atgtaagtag cttctagaag ctggaagtgg ctctcagttg acagccagcc 57300attaaggaaa ttaggatctc agttctgcaa ctataaggag ctgaattctg ccaagagacc 57360aatgtggaaa cagcagatcc ctccacagag acacaagctt actgataact ggtaggaatt 57420tctccaaaag tggagcttcc tcctactcca gtgttaatcc ctttctcaga ggagacggtc 57480ctcaaactaa ctaacttggc accaaaagtc ctatccagtg ttttctcatt atagtttttc 57540tatgcctcaa ctgtatatat ttacccagtt taggctgttt aaatgaataa aaaggaaatg 57600ccatagttat tctagccagt ttccaatctc tcttctcttt ttttgttttg tcaaataggg 57660cagataaggc atgagaattt ataactatga attactgtct tttcccaaac agaaatcacc 57720ctatcagctt acccattggg agaaaaacta aaatagctcc ccctgaaatt ttacttcctc 57780atttgggtct tgtgtgactg aaatctgtat acaatgccct agcaacaacg gtttttacag 57840cttgcctccc tagaacaaac ctaggagtct cagctgtttc aggaatgatt tcttaaaggt 57900aaagtgcctt tttcaaaaga aattattatt attttttttt aatttttttt ttgtgtgtgt 57960gtgagacaga gcctcactct gtcaccaggc tggagtgcag tggcacgatc tcagcacact 58020gcaacctctg cctcccaggt tcaagcgatt ctcctgcctc agcctcccaa gtagctggga 58080ctacaggcac gtgccaccaa gcccaggtaa tttttgtatt ttcagtagag atgggttttc 58140accatgttgg ccaggatggt ctcgatctct tgacctcgtg atccgttttt aaccaacatt 58200taaacagaaa tattcacagg cttaaagact gaaagttagt gatatcatca catttcccct 58260tcaaaatgct gaatttgtaa gcaaatttaa aagtttagaa tctacctttt aattgtctgc 58320tttcattttt ttgacagtgg ctttttttga tatggtgact attttgtcat gggtataaaa 58380ggataattca ttttgtgtta atctgaagac atctgaaata ctgtattcaa ctataagtac 58440ctttttttac atttataaga ttctttttca aaatttttat ttgaatagtt ttttgggaac 58500tactgaacta aactaggtgg tttttggtta catggataag ttatttagtg gtgatttctg 58560agactttggt gccacctgtc actcgagcag tgtacactgc accagtgtgt agtcttttat 58620ctctcacccc tcccactctt tcctctgagt ccccaaagtc cattatatta ttcttatgtc 58680tttgcatcct catagtttag ctcccactta tcagtgaaaa catacaatat ttgtttctcc 58740attcttgagt tacttcactt agaataatgg tctctggttc catcaaagtt gctgcaaatg 58800ccattatttt gtttcttttt atggctgagt aatattccat gagggatatt taccacattt 58860tccttatcca ctcatgggtt gatggacatt taggttggtt ccttattttt ggaattgcaa 58920attgtgctgc tataaacatg cgtgtgcatg tgtctttttc atataatgaa ttattttcct 58980ttgggtatat acccagtagt aggattgctg aattaaatag tagagttcta cttttagttc 59040tttaaggaat ctccatactg ttttccatag tgtttgtact agtttacatt cccaccagca 59100gtgtaaacat gttccctttt caccacatcc atgccaacat ctattatttt ttgatttttt 59160aataatggcc attcttgcag gagtaaggtg gtatctcatg gtggttttaa tttgcatttc 59220cctgatagtt agtgatattg aacttttttt catgtttgtt ggccatttgt atattttctt 59280ttcagaattg tctattcatg tccttataaa caccattatt tttaagaaga aactttacaa 59340aaatagaaca taaccagatt tataaagcat ctgggaactc agtcaattaa gaaatagctc 59400aagtaactga tgatgcttca cctgaaagaa ggcctggaga gaacagagat actgtcttca 59460aatatctgaa gagctaccat gggatgcaaa gattgagctt gatggtatga ctctgaaggg 59520catctctatg aatgaaggtt atgagagggt ataaggaatt aagagagact tttctaacaa 59580ttaaaaggtc ttttaggcca ggggtggtgg ctcacacctg taatcccagc acttttggag 59640gctgaggcag gcagatcacc ttagatcagg agttcgagac ccgcctggcc aacatggtga 59700aaccccattt ctactaaaca tacaaaaatt agctgggtgt ggtggcaggc acctgtaatc 59760ccagctactt gggaggctga gagaggagaa tcgcttgaac ctgggaggca gaggttgcag 59820tgagccaaga tcacaccact gcactccagc ctgggtgaca gaagatcaag attccgtctt 59880aaaaaatata aataaataaa taaataaata aatagtcttt aaaattgtat agaagaagta 59940gacttctgct tcctccaaca aaggattaac tgctatagga attgccctct ttccataaac 60000aactagaaag cagacaaaat atatgaaaca actgttttca gagatcggat gacagacagc 60060agaaaactgt agtccctgag tgaaggaaag aaaaaatgag ataagcccta tgattgctct 60120agtttgctgc ctggagccag tgtccaggcc cctctgaagg caggggagcc ctgatactga 60180actaggaaaa gacattgcaa gaaaagaaaa ctacaaacat ctctcgtgaa atgcttaaca 60240aaattagcaa ctaaaatcta gcaatatgtt aaaagtataa tacatcatga tcaagtgggg 60300tttattcaag aaacacaggt aagctcaaca ttcaaaaatc aggcaataac ctttactaca 60360taaataaact aaaaagaaaa aaacatatga tcatgtcaat ggatacagga aaaacttttg 60420acaaaattaa tacccattca tagttttaaa tggaaagaaa agctctcata aaaataggaa 60480tacaagatga cttcctcaac ctgacaaagg acatctacca aaaattcttc tgttagcata 60540atatttcatg atagaagact gattgctttt accttaagat ggcgaatgtg gggaggatgt 60600ctactctctc tacttttgtt ccacattgta ctggaggtca tagccagaga aacaagacta 60660gaaaaagaaa taaaagacat acagattgga aaggaagtaa aactgtcttt tttcacagat 60720aatgatcatg cttgtagaaa atcctgagga atctatcaaa aacctattaa aactgataag 60780tgagtgtagc aaagacacag gatacaaagt caatacacaa aatcaattat ttctatatac 60840taacaaaagc aattgtacat tgaaaaaaat taatagcatt tataatagca tcaaataata 60900ttaaaaactt ggaaataaat ttaacaaaac aagtacaagg tctatatact gaaaactata 60960caatattact actggagaaa ttaaagtaaa ccaaaataaa tggagacata ggccatgttt 61020atgaatcaga agactagatg ttaagataac cattctctcc aagttgatct atggattaaa 61080tgtaatcaca atcaaaatcc tggtaagctc tctaatagat actaaaaatc ttactcgaaa 61140agttataggg aaatgcaaag aatctacaat tgccaaaaca attctgaaaa ataagaacaa 61200aggttaaaaa tacaaaatta gccaggcatg gtggcgcatg cctgtaatcc cagctactct 61260ggaggctgag gcaggagaat tgcttgaacc cgggaggcag aggttgctgt gagctgagat 61320cgtgccattg cactccagcc tgggcaacaa gagtgaaact ccctctcaaa aaaaaaaaaa 61380aaaaaaaaaa aaaagaacaa aggtggactt aacctaccta atttcaatat ttactatata 61440tagtaattaa tacagtgtga tattggtaaa aggacagaca tatcagtcaa tggaacaaaa 61500tagagagtca aaaatagatt cacactgttg acaaagctac caaggtaatt ccatgcagaa 61560aggatagtat tttcaacaaa tagtgttggg acaattagat atccacatgg aaaaagtatg 61620aacctagaca cacacaaagt aacttatata ttaagaatta aaatgaaagg acttccaaaa 61680gaaaacagag gagaaaatct ttgtaacctt aagttaggca agtcttctta gataggacac 61740agaaagcaaa aaccatatca taaaaagata aaatggatgt catcaatatg gaaaactttt 61800gttctttgac tttgtttaaa aaacgaaaag tcaaaccaca gacagggaga aaacgtttgc 61860aaaatatata tctgataaag gacttgtatc cagtatataa ttacatattg ctactcatta 61920gtaagaagac aatccattta ataaaaggca agaagaagag acttgaacag atacataaca 61980gaagaagata tacagatggc cgatgagcac agtcacaaca tcattagtca tcagggaagt 62040acaaattaaa acgataatga gataccactg cacaccctct agaatggcta aaattaaaag 62100gtctgataaa catcaagtgt tggagaggat atgaagcaac tgaaactctc atatactgct 62160atacaaccca gaaatcctag acatttacca aacagaaatt ttaaaaaatt taaaaatata 62220taaagactca tacacaaatg ttcatagcag cttgcttcat aataccaaac ctggcattct 62280aaattttcat cagttggcgg tggtatattt atacaatgaa atactgcaaa gctatagaaa 62340ggaatggact actaataata cacaagaaca tagataaatt tcaaaagcat tatgctaagt 62400gaaacaatcc aggcacaaga agaatacaca ttatacaatt tcatgtatat gaaatttgag 62460aaaaagcaaa actattttaa gtagattcat ggttatccat gggatggggg aaaggaatca 62520gctgaaaagc gaactatttt ggcttataaa aatgttctcg atcttgattg tggtggtggt 62580tacgtgacta tatatattcg ttaaaatcac caaactctaa actgaaaatg attgggtttt 62640attatttatt aattatacct ccataaagct gattgttttt atcttttatt tttattttat 62700ttcaatagtt tttggggaac agatggtttt cggttacatg gatgagttct ttagtggtga 62760tttctgagat tttgatgcac ctgtcacccg agcaatgtcc actgtaccca atgtgtagtc 62820ttttatcctt catccacctc tctctcactc ttccccccaa gtacccaagt ccattatatc 62880attcttatga ctttgtggcc tcataaaagc tgattgtttt taaatacaca catacacaca 62940taaaagagaa cttccagtga caggaagtgt tcaagaatgc tctatttagt aaagacagaa 63000tcacaaaacc atcagaggta ttgttgagtg gattcttgtg gtctataaat acctccatgg 63060acacccaggt tagcaacctg ttggagttta cgtgggacaa tagcatcatc acaacagtca 63120gcctagagaa atttacatcc caagttgtgt cagtagcaag tccctatcaa tagcaactca 63180ggctttgtga ggtctagctg gctagaaatt tcccacttgg ccttgcccat gcaacattgt 63240gtaatattct tagcaccatc tggctagccg atttaggcat caacatcttc aagacttctt 63300ctcctcctcc ttataaacct tgctttcaga aaaggattag aaactcttcc aatcacaaaa 63360tgattgctaa aactaaatat attacccctc ccaatggtat tttttggtta gccaggatag 63420agatataagt gaaaaatcta tttccagtgt tagaatttaa ggcacagtga gaaagggaag 63480gcatatactt tttgaatgca agaaacttct tcccaatccc cctgaaattg catcatttga 63540gtaactatct cttccatata taaagtcaca acaatttctc tctcagtccc agaactttga 63600agccttttca aactttcctt cttttggtat ctaggaggaa tacatttttg aagattgttc 63660ttggtgtctt tcaggaacca acacaatgga gagggaagag agtgaacaga ccaagaaaag 63720gtaaatcctg accctgagac attgatgaga gagaggtata atccccagag tgcctgttac 63780ttgaataggc ttatgcctaa catatgttga gacctcagca aacctgaact aatggagagg 63840gagaggaaaa taaaactagt taagaactgg aagaaaataa cctgataatg gatgacaggg 63900tatccaatgc acaatgccca gaaagcatga caagctctgt catggtcaag taaaagtcaa 63960taccaaagac ttcagaggtg gtgaacatgg gcttcatctt atctgccaca gtaaccccag 64020tacctggcac agtgcctaga ttagtgggca tcctacatgt gtggaatgaa taaatgaaga 64080agtggggaat gataacatgt ttgcttcagc ctgagcatct tagtatttgc tatggccctg 64140tttagatgtt cttctgccac ttctttacct cattcttcag atcttgcctc aagcagcact 64200ttcttaaaaa ccctttccca aactagaaaa tgtcaacttg ttacagtgtc atgtggatcc 64260cttggctttt tcttaataac accagattat gcttacatat ttgtgtaatt atcttattaa 64320actctataaa ctagacttaa ctaaatccta tgaagagcag agaccatacc agttaagctc 64380atcattgtgc tgctagcact tagcatggtg cctggcatat agcaggttct caataaatgt 64440tgaaagaatg attgatgcat gatgaataca taaaagttcg tggtgatcag tcctttcaca 64500acgtgaagct atcagatagt ctgtacctct atccctcctg agaaattaag ctctcaggaa 64560tatcaaggct ctgactgcat acccatagga tcaaagcaac cctcagtcac aagcctggtt 64620tcagagatag ggtcataacc cccagggtgc agagacaacc gagagtaccc agcactaatc 64680cagatatacc agccactgtg attctagcaa caaaactaat aattccgggc acccttggac 64740aatgagaaag ggtgctgaaa tcctgcctac cctgtcacac tcagtttcag aaatggtctg 64800gaagagcctg cagagggcag gcagcagaga accggcagag ggcatgggaa gggccaggca 64860gaaataaagg gtagctcttg aagcatagat gacagtgtag accgtggttc ttttctcttg 64920ctttctccac ctttctcttc aatagtttgt ttctcctcat tgctgttcca atggcaacct 64980ctattctgcc ctatcattga aatctagaaa aagaaagtag ctcaaatgtg aaatatcacc 65040taatcttttc ttctatttct ccagagaaaa aatccatata cctgaaagat ctgatgaagc 65100ccagcgtgtt tttaaaagtt cgaagacatc ttcatgcgac aaaagtgata catgttttta 65160attaaagagt aaagcccata caagtattca ttttttctac cctttccttt gtaagttcct 65220gggcaacctt tttgatttct tccagaaggc aaaaagacat taccatgagt aataaggggg 65280ctccaggact ccctctaagt ggaatagcct ccctgtaact ccagctctgc tccgtatgcc 65340aagaggagac tttaattctc ttactgcttc ttttcacttc agagcacact tatgggccaa 65400gcccagctta atggctcatg acctggaaat aaaatttagg accaatacct cctccagatc 65460agattcttct cttaatttca tagattgtgt ttttttttta aatagacctc tcaatttctg 65520gaaaactgcc ttttatctgc ccagaattct aagctggtgc cccactgaat tttgtgtgta 65580cctgtgacta aacaactacc tcctcagtct gggtgggact tatgtattta tgaccttata 65640gtgttaatat cttgaaacat agagatctat gtactgtaat agtgtgatta ctatgctcta 65700gagaaaagtc tacccctgct aaggagttct catccctctg tcagggtcag taaggaaaac 65760ggtggcctag ggtacaggca acaatgagca gaccaaccta aatttgggga aattaggaga 65820ggcagagata gaacctggag ccacttctat ctgggctgtt gctaatattg aggaggcttg 65880ccccacccaa caagccatag tggagagaac tgaataaaca ggaaaatgcc agagcttgtg 65940aaccctgttt ctcttgaaga actgactagt gagatggcct ggggaagctg tgaaagaacc 66000aaaagagatc acaatactca aaagagagag agagagaaaa aagagagatc ttgatccaca 66060gaaatacatg aaatgtctgg tctgtccacc ccatcaacaa gtcttgaaac aagcaacaga 66120tggatagtct gtccaaatgg acataagaca gacagcagtt tccctggtgg tcagggaggg 66180gttttggtga tacccaagtt attgggatgt catcttcctg gaagcagagc tggggaggga 66240gagccatcac cttgataatg ggatgaatgg aaggaggctt aggactttcc actcctggct 66300gagagaggaa gagctgcaac ggaattagga agaccaagac acagatcacc cggggcttac 66360ttagcctaca gatgtcctac gggaacgtgg gctggcccag catagggcta gcaaatttga 66420gttggatgat tgtttttgct caaggcaacc agaggaaact tgcatacaga gacagatata 66480ctgggagaaa tgactttgaa aacctggctc taaggtggga tcactaaggg atggggcagt 66540ctctgcccaa acataaagag aactctgggg agcctgagcc acaaaaatgt tcctttattt 66600tatgtaaacc ctcaagggtt atagactgcc atgctagaca agcttgtcca tgtaatattc 66660ccatgttttt accctgcccc tgccttgatt agactcctag cacctggcta gtttctaaca 66720tgttttgtgc agcacagttt ttaataaatg cttgttacat tcatttaaaa gtctacattt 66780tctgctttgg cttcaagagt actactcaac ccttgtggtc tgatgttccc tgctctgtcc 66840tctgaatgta cttcctttct ctttacatct ctatggctag aagcctctca cgcatcctgt 66900atcttctcct cctccctttt ccctaccatt atttgagaaa ggaggcttgt atacttctat 66960atgtttatct cagtaataag tcataaaaaa tcaagtaaga atggttgttt ttgaggacaa 67020ctaagaaatc tggaataagg aagggaagct tacttttgag tttgtaacct gtagtgtgta 67080attttttaat tatgtactta catgtacatt aaacaaaagc ttaatgtaaa aatattcctt 67140gaaaacacca tgattataaa ataaatgcat atatacacat acagcatgtg agaggagcca 67200ggaaaactct ggaaaaaaga aaattaccta gactctgtga gggcaggaat gtgtttaatt 67260tctctccaat ggatcctcag acaactaaga tagttgtcta ttctattgtc catctttttg 67320tcttttgttg tatttcttaa agattccctc aactttatct tctaacttct gttgtatttt 67380tatttctgct atcatgtatt cttttcagaa ttcttttttg ttctctcaaa acatatctgt 67440ttaaagattg aatgaaatat taacatgccc tttggtgaga acatccctcc tttgtatatt 67500aaattctctg aactgctgta ttctaagact aggggaaaga aaaagaaggt tgaaagaggt 67560cattaggcag aatagtacta gctaacatta tttcacattt accatatacc cgtcactcat 67620ctaaaccttt aaactcatta tcctatttaa tcctcacaat gaccctgtga cgtaggtaat 67680ggaatattat gcccattatg ctgatgagaa aatataaaca cagagataag tcagagtaat 67740ttacccaaca ttgttaactt tgtaagtggc agagctttgt aacaggcaga ggttggaaca 67800gtttggaggg ctcagaagaa gacaggaaga tgtaggaaag tttggaactt cccagagcct 67860tgttgaatgg ctttgaccaa aatgctgata gtaatatgga caatgaaata caggctgagg 67920tggtctcaga tagagaagag gaacttgttg ggaactggaa taaaggtgac tcttgctatg 67980ttttagcaaa gacactggtg g 68001 298 20 DNA Artificial Sequence AntisenseOligonucleotide 298 accaaaagga gtatttgcga 20 299 20 DNA ArtificialSequence Antisense Oligonucleotide 299 cattcccaag gaacacagaa 20 300 20DNA Artificial Sequence Antisense Oligonucleotide 300 actgtagctccaaaaagaga 20 301 20 DNA Artificial Sequence Antisense Oligonucleotide301 ctgtcacaaa tgcctgtcca 20 302 20 DNA Artificial Sequence AntisenseOligonucleotide 302 tcagtcccat agtgctgtca 20 303 20 DNA ArtificialSequence Antisense Oligonucleotide 303 ctgttacagc agcagagaag 20 304 20DNA Artificial Sequence Antisense Oligonucleotide 304 tccctgttacagcagcagag 20 305 20 DNA Artificial Sequence Antisense Oligonucleotide305 atctggaaat gaccccactc 20 306 20 DNA Artificial Sequence AntisenseOligonucleotide 306 gtgacctaat atctggaaat 20 307 20 DNA ArtificialSequence Antisense Oligonucleotide 307 cattttggct gcttctgctg 20 308 20DNA Artificial Sequence Antisense Oligonucleotide 308 ggaacttacaaaggaaaggg 20 309 20 DNA Artificial Sequence Antisense Oligonucleotide309 aaaaaggttg cccaggaact 20 310 20 DNA Artificial Sequence AntisenseOligonucleotide 310 tgccttctgg aagaaatcaa 20 311 20 DNA ArtificialSequence Antisense Oligonucleotide 311 tttttgcctt ctggaagaaa 20 312 20DNA Artificial Sequence Antisense Oligonucleotide 312 ctattccacttagagggagt 20 313 20 DNA Artificial Sequence Antisense Oligonucleotide313 tctgatctgg aggaggtatt 20 314 20 DNA Artificial Sequence AntisenseOligonucleotide 314 agaaattgag aggtctattt 20 315 20 DNA ArtificialSequence Antisense Oligonucleotide 315 caccagctta gaattctggg 20 316 20DNA Artificial Sequence Antisense Oligonucleotide 316 aggtagttgtttagtcacag 20 317 20 DNA Artificial Sequence Antisense Oligonucleotide317 ccagactgag gaggtagttg 20 318 20 DNA Artificial Sequence AntisenseOligonucleotide 318 cagtacatag atctctatgt 20 319 20 DNA ArtificialSequence Antisense Oligonucleotide 319 ttacagtaca tagatctcta 20 320 20DNA Artificial Sequence Antisense Oligonucleotide 320 gatgagaactccttagcagg 20 321 20 DNA Artificial Sequence Antisense Oligonucleotide321 tagcaacagc ccagatagaa 20 322 20 DNA Artificial Sequence AntisenseOligonucleotide 322 tctgttgctt gtttcaagac 20 323 20 DNA ArtificialSequence Antisense Oligonucleotide 323 tccatttgga cagactatcc 20 324 20DNA Artificial Sequence Antisense Oligonucleotide 324 gggaaactgctgtctgtctt 20 325 20 DNA Artificial Sequence Antisense Oligonucleotide325 tgcttccagg aagatgacat 20 326 20 DNA Artificial Sequence AntisenseOligonucleotide 326 attcatccca ttatcaaggt 20 327 20 DNA ArtificialSequence Antisense Oligonucleotide 327 agccaggagt ggaaagtcct 20 328 20DNA Artificial Sequence Antisense Oligonucleotide 328 cttcctaattccgttgcagc 20 329 20 DNA Artificial Sequence Antisense Oligonucleotide329 catctgtagg ctaagtaagc 20 330 20 DNA Artificial Sequence AntisenseOligonucleotide 330 cccgtaggac atctgtaggc 20 331 20 DNA ArtificialSequence Antisense Oligonucleotide 331 gccctatgct gggccagccc 20 332 20DNA Artificial Sequence Antisense Oligonucleotide 332 gtctctgtatgcaagtttcc 20 333 20 DNA Artificial Sequence Antisense Oligonucleotide333 ccagtatatc tgtctctgta 20 334 20 DNA Artificial Sequence AntisenseOligonucleotide 334 ccaggttttc aaagtcattt 20 335 20 DNA ArtificialSequence Antisense Oligonucleotide 335 agccaggttt tcaaagtcat 20 336 20DNA Artificial Sequence Antisense Oligonucleotide 336 cccttagtgatcccacctta 20 337 20 DNA Artificial Sequence Antisense Oligonucleotide337 ctgccccatc ccttagtgat 20 338 20 DNA Artificial Sequence AntisenseOligonucleotide 338 tttatgtttg ggcagagact 20 339 20 DNA ArtificialSequence Antisense Oligonucleotide 339 catggcagtc tataaccctt 20 340 20DNA Artificial Sequence Antisense Oligonucleotide 340 tagcatggcagtctataacc 20 341 20 DNA Artificial Sequence Antisense Oligonucleotide341 tctagcatgg cagtctataa 20 342 20 DNA Artificial Sequence AntisenseOligonucleotide 342 ttgtctagca tggcagtcta 20 343 20 DNA ArtificialSequence Antisense Oligonucleotide 343 aagcttgtct agcatggcag 20 344 20DNA Artificial Sequence Antisense Oligonucleotide 344 acatggacaagcttgtctag 20 345 20 DNA Artificial Sequence Antisense Oligonucleotide345 ttacatggac aagcttgtct 20 346 20 DNA Artificial Sequence AntisenseOligonucleotide 346 gaatattaca tggacaagct 20 347 20 DNA ArtificialSequence Antisense Oligonucleotide 347 aactagccag gtgctaggag 20 348 20DNA Artificial Sequence Antisense Oligonucleotide 348 aattattactcaccactggg 20 349 20 DNA Artificial Sequence Antisense Oligonucleotide349 taatatttag ggaagcatga 20 350 20 DNA Artificial Sequence AntisenseOligonucleotide 350 ggaccctggg ccagttattg 20 351 20 DNA ArtificialSequence Antisense Oligonucleotide 351 caaacatacc tgtcacaaat 20 352 20DNA Artificial Sequence Antisense Oligonucleotide 352 gtgatatcaattgatggcat 20 353 20 DNA Artificial Sequence Antisense Oligonucleotide353 tgctacatct actcagtgtc 20 354 20 DNA Artificial Sequence AntisenseOligonucleotide 354 tggaaactct tgccttcgga 20 355 20 DNA ArtificialSequence Antisense Oligonucleotide 355 ccatccacat tgtagcatgt 20 356 20DNA Artificial Sequence Antisense Oligonucleotide 356 tcaggatggtatggccatac 20 357 20 DNA Artificial Sequence Antisense Oligonucleotide357 tcccatagtg ctagagtcga 20 358 20 DNA Artificial Sequence AntisenseOligonucleotide 358 aggttcttac cagagagcag 20 359 20 DNA ArtificialSequence Antisense Oligonucleotide 359 cagaggagca gcacctaaaa 20 360 20DNA Artificial Sequence Antisense Oligonucleotide 360 gaccacataccaagcactga 20 361 20 DNA Artificial Sequence Antisense Oligonucleotide361 atctttcaga aacccaagca 20 362 20 DNA Artificial Sequence AntisenseOligonucleotide 362 gagtcaccaa agatttacaa 20 363 20 DNA ArtificialSequence Antisense Oligonucleotide 363 ctgaagttag ctgaaagcag 20 364 20DNA Artificial Sequence Antisense Oligonucleotide 364 acagctttacctatagagaa 20 365 20 DNA Artificial Sequence Antisense Oligonucleotide365 tcctcaagct ctacaaatga 20 366 20 DNA Artificial Sequence AntisenseOligonucleotide 366 gactcactca ccacatttat 20 367 20 DNA ArtificialSequence Antisense Oligonucleotide 367 agtgatagca aggcttctct 20 368 20DNA Artificial Sequence Antisense Oligonucleotide 368 cttggagagaatggttatct 20 369 20 DNA Artificial Sequence Antisense Oligonucleotide369 gaagatgttg atgcctaaat 20 370 20 DNA Artificial Sequence AntisenseOligonucleotide 370 gtgttggttc ctgaaagaca 20 371 20 DNA ArtificialSequence Antisense Oligonucleotide 371 caggatttac cttttcttgg 20 372 20DNA Artificial Sequence Antisense Oligonucleotide 372 agggcagaatagaggttgcc 20 373 20 DNA Artificial Sequence Antisense Oligonucleotide373 tttttctctg gagaaataga 20 374 20 DNA Artificial Sequence AntisenseOligonucleotide 374 gttactcagt cccatagtgc 20 375 20 DNA ArtificialSequence Antisense Oligonucleotide 375 caaagagaat gttactcagt 20 376 20DNA Artificial Sequence Antisense Oligonucleotide 376 ccatcacaaagagaatgtta 20 377 20 DNA Artificial Sequence Antisense Oligonucleotide377 ggaaggccat cacaaagaga 20 378 20 DNA Artificial Sequence AntisenseOligonucleotide 378 gagcaggaag gccatcacaa 20 379 20 DNA ArtificialSequence Antisense Oligonucleotide 379 ccagagagca ggaaggccat 20 380 20DNA Artificial Sequence Antisense Oligonucleotide 380 aaataagcttgaatcttcag 20 381 20 DNA Artificial Sequence Antisense Oligonucleotide381 agtctcattg aaataagctt 20 382 20 DNA Artificial Sequence AntisenseOligonucleotide 382 aggtctgcag tctcattgaa 20 383 20 DNA ArtificialSequence Antisense Oligonucleotide 383 ctactagctc actcaggctt 20 384 20DNA Artificial Sequence Antisense Oligonucleotide 384 aaatactactagctcactca 20 385 20 DNA Artificial Sequence Antisense Oligonucleotide385 ctgccaaaat actactagct 20 386 20 DNA Artificial Sequence AntisenseOligonucleotide 386 ttcagaacca agttttcctg 20 387 20 DNA ArtificialSequence Antisense Oligonucleotide 387 cctcattcag aaccaagttt 20 388 20DNA Artificial Sequence Antisense Oligonucleotide 388 gtatacctcattcagaacca 20 389 20 DNA Artificial Sequence Antisense Oligonucleotide389 gcctaagtat acctcattca 20 390 20 DNA Artificial Sequence AntisenseOligonucleotide 390 ctctttgcct aagtatacct 20 391 20 DNA ArtificialSequence Antisense Oligonucleotide 391 cccatatact tggaatgaac 20 392 20DNA Artificial Sequence Antisense Oligonucleotide 392 cttgtgcggcccatatactt 20 393 20 DNA Artificial Sequence Antisense Oligonucleotide393 atcaaaactt gtgcggccca 20 394 20 DNA Artificial Sequence AntisenseOligonucleotide 394 cccttgtcct tgatctgaag 20 395 20 DNA ArtificialSequence Antisense Oligonucleotide 395 acaagccctt gtccttgatc 20 396 20DNA Artificial Sequence Antisense Oligonucleotide 396 ttgatacaagcccttgtcct 20 397 20 DNA Artificial Sequence Antisense Oligonucleotide397 atacattgat acaagccctt 20 398 20 DNA Artificial Sequence AntisenseOligonucleotide 398 tggatgatac attgatacaa 20 399 20 DNA ArtificialSequence Antisense Oligonucleotide 399 gaattcatct ggtggatgcg 20 400 20DNA Artificial Sequence Antisense Oligonucleotide 400 gttcagaattcatctggtgg 20 401 20 DNA Artificial Sequence Antisense Oligonucleotide401 tgacagttca gaattcatct 20 402 20 DNA Artificial Sequence AntisenseOligonucleotide 402 agcactgaca gttcagaatt 20 403 20 DNA ArtificialSequence Antisense Oligonucleotide 403 tagcaagcac tgacagttca 20 404 20DNA Artificial Sequence Antisense Oligonucleotide 404 tgaagttagcaagcactgac 20 405 20 DNA Artificial Sequence Antisense Oligonucleotide405 ttgactgaag ttagcaagca 20 406 20 DNA Artificial Sequence AntisenseOligonucleotide 406 ctatttcagg ttgactgaag 20 407 20 DNA ArtificialSequence Antisense Oligonucleotide 407 tctgttatat tagaaattgg 20 408 20DNA Artificial Sequence Antisense Oligonucleotide 408 gcaggtcaaatttatgtaca 20 409 20 DNA Artificial Sequence Antisense Oligonucleotide409 gtatagatga gcaggtcaaa 20 410 20 DNA Artificial Sequence AntisenseOligonucleotide 410 gggtaaccgt gtatagatga 20 411 20 DNA ArtificialSequence Antisense Oligonucleotide 411 aggttctggg taaccgtgta 20 412 20DNA Artificial Sequence Antisense Oligonucleotide 412 tagcaaaacactcatcttct 20 413 20 DNA Artificial Sequence Antisense Oligonucleotide413 gttcttagca aaacactcat 20 414 20 DNA Artificial Sequence AntisenseOligonucleotide 414 attcttggtt cttagcaaaa 20 415 20 DNA ArtificialSequence Antisense Oligonucleotide 415 gatagttgaa ttcttggttc 20 416 20DNA Artificial Sequence Antisense Oligonucleotide 416 accatcatactcgatagttg 20 417 20 DNA Artificial Sequence Antisense Oligonucleotide417 atcttgagat ttctgcataa 20 418 20 DNA Artificial Sequence AntisenseOligonucleotide 418 acattatctt gagatttctg 20 419 20 DNA ArtificialSequence Antisense Oligonucleotide 419 cgtacagttc tgtgacatta 20 420 20DNA Artificial Sequence Antisense Oligonucleotide 420 agacaagctgatggaaacgt 20 421 20 DNA Artificial Sequence Antisense Oligonucleotide421 gaaacagaca agctgatgga 20 422 20 DNA Artificial Sequence AntisenseOligonucleotide 422 ggaatgaaac agacaagctg 20 423 20 DNA ArtificialSequence Antisense Oligonucleotide 423 catcagggaa tgaaacagac 20 424 20DNA Artificial Sequence Antisense Oligonucleotide 424 cgtaacatcagggaatgaaa 20 425 20 DNA Artificial Sequence Antisense Oligonucleotide425 agctctatag agaaaggtga 20 426 20 DNA Artificial Sequence AntisenseOligonucleotide 426 cctcaagctc tatagagaaa 20 427 20 DNA ArtificialSequence Antisense Oligonucleotide 427 ggaggctgag ggtcctcaag 20 428 20DNA Artificial Sequence Antisense Oligonucleotide 428 agtacagctgtaatccaagg 20 429 20 DNA Artificial Sequence Antisense Oligonucleotide429 ttggaagtac agctgtaatc 20 430 20 DNA Artificial Sequence AntisenseOligonucleotide 430 ataataactg ttggaagtac 20 431 20 DNA ArtificialSequence Antisense Oligonucleotide 431 catcacacat ataataactg 20 432 20DNA Artificial Sequence Antisense Oligonucleotide 432 tccatttccatagaattaga 20 433 20 DNA Artificial Sequence Antisense Oligonucleotide433 tcttcttcca tttccataga 20 434 20 DNA Artificial Sequence AntisenseOligonucleotide 434 atttataaga gttgcgaggc 20 435 20 DNA ArtificialSequence Antisense Oligonucleotide 435 ttggttccac atttataaga 20 436 20DNA Artificial Sequence Antisense Oligonucleotide 436 ctctccattgtgttggttcc 20 437 20 DNA Artificial Sequence Antisense Oligonucleotide437 cttccctctc cattgtgttg 20 438 20 DNA Artificial Sequence AntisenseOligonucleotide 438 tggtctgttc actctcttcc 20 439 20 DNA ArtificialSequence Antisense Oligonucleotide 439 ttcatcagat ctttcaggta 20 440 20DNA Artificial Sequence Antisense Oligonucleotide 440 atcacttttgtcgcatgaag 20 441 20 DNA Artificial Sequence Antisense Oligonucleotide441 gctttactct ttaattaaaa 20 442 20 DNA Artificial Sequence AntisenseOligonucleotide 442 gtatgggctt tactctttaa 20 443 20 DNA ArtificialSequence Antisense Oligonucleotide 443 atacttgtat gggctttact 20 444 20DNA Artificial Sequence Antisense Oligonucleotide 444 aatgaatacttgtatgggct 20

What is claimed is:
 1. A method for treating airway hyperresponsivenessor pulmonary inflammation in an individual in need thereof, comprisingadministering to said individual an antisense compound 8 to 30nucleobases in length targeted to a nucleic acid molecule encoding ahuman B7 protein to said individual.
 2. The method of claim 1, whereinsaid antisense compound is an antisense oligonucleotide.
 3. The methodof claim 2, wherein at least one covalent linkage of said antisensecompound is a modified covalent linkage.
 4. The method of claim 2,wherein at least one nucleotide of said antisense compound has amodified sugar moiety.
 5. The method of claim 2, wherein at least onenucleotide of said antisense compound has a modified nucleobase.
 6. Themethod of claim 1, wherein said human B7 protein is human B7-1 protein.7. The method of claim 1, wherein said human B7 protein is human B7-2protein.
 8. The method of claim 1, wherein said human B7 protein is bothhuman B7.1 and human B7.2 protein.
 9. The method of claim 1, furthercomprising administering an anti-asthma medication to said individual.10. The method of claim 1 wherein said antisense compound comprises atleast one lipophilic moiety which oligonucleotide is aerosolized andinhaled by said individual.
 12. The method of claim 1, wherein saidoligonucleotide is administered intranasally, intrapulmonarily orintratracheally.
 13. The method of claim 1, wherein said airwayhyperresponsiveness or pulmonary inflammation is associated with asthma.14. A pharmaceutical composition comprising an antisense oligonucleotidetargeted to nucleic acid encoding human B7.1 or B7.2 in a formulationsuitable for intranasal, intrapulmonary or intratracheal administration.15. The pharmaceutical composition of claim 14, wherein said compositionis in a metered dose inhaler or nebulizer.
 16. An RNA compound 8 to 80nucleobases in length targeted to a nucleic acid molecule encoding ahuman B7 protein, wherein said compound specifically hybridizes withsaid nucleic acid molecule encoding human B7 protein and inhibits theexpression of human B7 protein.
 17. The RNA compound of claim 16 whereinsaid B7 protein is B7.1.
 18. The RNA compound of claim 16 wherein saidB7 protein is B7.2.